Apparatus and method for cell disruption

ABSTRACT

An apparatus for disrupting cells or viruses comprises a container having a chamber for holding the cells or viruses. The container includes at least one flexible wall defining the chamber. The apparatus also includes a transducer for impacting an external surface of the flexible wall to generate pressure waves in the chamber. The apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall. The apparatus may also include beads in the chamber for rupturing the cells or viruses.

RELATED APPLICATION INFORMATION

This application is a continuation of U.S. patent application no.10/759,741, filed Jan. 15, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/584,327, filed May 30, 2000 (abandoned). Theseapplications are incorporated by reference herein for all purposes.

FIELD OF THE INVENTION

The present invention relates to an apparatus and method for rapidlydisrupting cells or viruses.

BACKGROUND OF THE INVENTION

The extraction of nucleic acid from cells or viruses is a necessary taskfor many applications in the fields of molecular biology and biomedicaldiagnostics. Once released from the cells, the nucleic acid may be usedfor genetic analysis, e.g., sequencing, pathogen identification andquantification, nucleic acid mutation analysis, genome analysis, geneexpression studies, pharmacological monitoring, storing of DNA librariesfor drug discovery, etc. The genetic analysis typically involves nucleicacid amplification and detection using known techniques. For example,known polynucleotide amplification reactions include polymerase chainreaction (PCR), ligase chain reaction (LCR), QB replicase amplification(QBR), self-sustained sequence replication (3SR), strand-displacementamplification (SDA), “branched chain” DNA amplification, ligationactivated transcription (LAT), nucleic acid sequence-based amplification(NASBA), repair chain reaction (RCR), and cycling probe reaction (CPR).

The extraction of nucleic acids from cells or viruses is generallyperformed by physical or chemical methods. Chemical methods typicallyemploy lysing agents (e.g., detergents, enzymes, or strong organics) todisrupt the cells and release the nucleic acid, followed by treatment ofthe extract with chaotropic salts to denature any contaminating orpotentially interfering proteins. Such chemical methods are described inU.S. Pat. No. 5,652,141 to Henco et al. and U.S. Pat. No. 5,856,174 toLipshutz et al. One disadvantage to the use of harsh chemicals fordisrupting cells is that the chemicals are inhibitory to subsequentamplification of the nucleic acid. In using chemical disruption methods,therefore, it is typically necessary to purify the nucleic acid releasedfrom the cells before proceeding with further analysis. Suchpurification steps are time consuming, expensive, and reduce the amountof nucleic acid recovered for analysis.

Physical methods for disrupting cells often do not require harshchemicals that are inhibitory to nucleic acid amplification (e.g., PCR).These physical methods, however, also have their disadvantages. Forexample, one physical method for disrupting cells involves placing thecells in a solution and heating the solution to a boil to break open thecell walls. Unfortunately, the heat will often denature proteins andcause the proteins to stick to the released nucleic acid. The proteinsthen interfere with subsequent attempts to amplify the nucleic acid.Another physical method is freeze thawing in which the cells arerepeatedly frozen and thawed until the cells walls are broken.Unfortunately, freeze thawing often fails to break open many structures,most notably certain spores and viruses that have extremely tough outerlayers.

Another physical method for disrupting cells is the use of a pressureinstrument. With this method, a solution of mycobacterial microorganismsis passed through a very small diameter hole under high pressure. Duringpassage through the hole, the mycobacteria are broken open by themechanical forces and their internal contents are spilled into solution.Such a system, however, is large, expensive and requires a coolingsystem to prevent excessive heat from building up and damaging thecontents of the lysed cells. Moreover, the instrument needs to becleaned and decontaminated between runs and a large containment systemis required when infectious material is handled. A further disadvantageto this system is that the solution must contain only particles havingsubstantially the same size, so that it may not be used to process manyuntreated clinical or biological specimens.

It is also known that cells can be lysed by subjecting the cells toultrasonic agitation. Typically, the cells are disrupted by placing anultrasonic probe directly into a volume of liquid containing the cells.Since the probe is in direct contact with a sample liquid, crosscontamination and cavitation-induced foaming present seriouscomplications.

Another method for cell disruption is disclosed by Murphy et al. in U.S.Pat. No. 5,374,522. According to the method, solutions or suspensions ofcells are placed in a container with small beads. The container is thenplaced in an ultrasound bath until the cells disrupt, releasing theircellular components. This method has several disadvantages. First, thedistribution of ultrasonic energy in the bath is not uniform, so that atechnician must locate a high energy area within the bath and place thecontainer into that area. The non-uniform distribution of ultrasonicenergy also produces inconsistent results. Second, the ultrasound bathdoes not focus energy into the container so that the disruption of thecells often takes several minutes to complete, a relatively long periodof time when compared to the method of the present invention. Third, itis not practical to carry an ultrasound bath into the field for use inbiowarfare detection, forensic analysis, or on-site testing ofenvironmental samples.

SUMMARY

The present invention overcomes the disadvantages of the prior art byproviding an improved apparatus and method for disrupting cells orviruses. In contrast to the prior art methods described above, thepresent invention provides for the rapid and effective disruption ofcells or viruses, including tough spores, without requiring the use ofharsh chemicals. The disruption of the cells or viruses can often becompleted in 5 to 10 seconds. In addition, the apparatus and method ofthe present invention provide for highly consistent and repeatable lysisof cells or viruses, so that consistent results are achieved from oneuse of the apparatus to the next.

According to a first embodiment, the apparatus comprises a containerhaving a chamber for holding the cells or viruses. The containerincludes at least one flexible wall defining the chamber. The apparatusalso includes a transducer, such as an ultrasonic horn, for impacting anexternal surface of the flexible wall to generate dynamic pressurepulses or pressure waves in the chamber. The apparatus also includes apressure source for increasing the pressure in the chamber. Thepressurization of the chamber ensures effective coupling between thetransducer and the flexible wall. The apparatus may also include beadsin the chamber for rupturing the cells or viruses.

In operation, the cells or viruses to be disrupted are placed in thechamber of the container. A liquid is also placed in the chamber. In oneembodiment, the cells or viruses are placed in the chamber by capturingthe cells or viruses on at least one filter positioned in the chamber.In this embodiment, the liquid placed in the chamber is usually a lysisbuffer added to the chamber after the cells or viruses have beencaptured. In an alternative embodiment, the liquid placed in the chambercontains the cells or viruses to be disrupted (e.g., the liquid is asample containing the cells or viruses) so that the liquid and cells areplaced in the chamber simultaneously. In either embodiment, thetransducer is placed against the external surface of the flexible wall,and the static pressure in the chamber is increased. Disruption of thecells is accomplished by impacting the flexible wall with the transducerto generate dynamic pressure pulses or pressure waves in the chamber.Beads may also be agitated in the chamber to rupture the cells orviruses.

A greater understanding of the invention may be gained by consideringthe following detailed description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an isometric view of a cartridge for analyzing a fluid sampleaccording to a first embodiment of the invention.

FIG. 2 is a lower isometric view of the cartridge of FIG. 1.

FIG. 3 is an exploded view of the cartridge of FIG. 1.

FIG. 4 is another exploded view of the cartridge of FIG. 1.

FIG. 5 is a partially cut away view of an ultrasonic horn coupled to awall of a lysing chamber formed in the cartridge of FIG. 1.

FIG. 6 is an exploded view of a filter stack positioned in the lysingchamber of the cartridge of FIG. 1.

FIG. 7 is a top plan view of the cartridge of FIG. 1.

FIG. 8 is a bottom plan view of the cartridge of FIG. 1.

FIG. 9 is a schematic block diagram of the cartridge of FIG. 1.

FIG. 10 is an isometric view of an instrument into which the cartridgeof FIG. 1 is placed for processing.

FIG. 11 is an isometric view of the cartridge of FIG. 1 in theinstrument of FIG. 10.

FIG. 12 is a partially cut-away view of the cartridge of FIG. 1 in theinstrument of FIG. 10.

FIG. 13 is a schematic, plan view of optical sensors positioned todetect liquid levels in the cartridge of FIG. 1.

FIG. 14 is a partially cut away, schematic, side view of a slottedoptical sensor positioned to detect the liquid level in a sensor chamberof the cartridge of FIG. 1.

FIG. 15A is a cross-sectional view of a portion of the body of thecartridge of FIG. 1 illustrating two different types of valves in thecartridge.

FIG. 15B is a cross-sectional view of the valves of FIG. 15A in a closedposition.

FIG. 16A is another cross-sectional view of one of the valves of FIG.15A in an open position.

FIG. 16B is a cross-sectional view of the valve of FIG. 16A in a closedposition.

FIGS. 17-19 illustrate a valve actuation system for opening and closingthe valves of FIG. 15A.

FIG. 20 is a cross sectional view of alternative valve actuators foropening and closing the valves in the cartridge of FIG. 1. FIG. 20 alsoshows a pressure delivery nozzle sealed to a pressure port formed in thecartridge of FIG. 1.

FIG. 21 is a partially exploded, isometric view of a reaction vessel ofthe cartridge of FIG. 1.

FIG. 22 is a front view of the vessel of FIG. 21.

FIG. 23 is a side view of the vessel of FIG. 21 inserted between twoheater plates.

FIG. 24 is a front view of one of the heater plates of FIG. 23.

FIG. 25 is a front view of an alternative reaction vessel according tothe present invention.

FIG. 26 is a front view of another reaction vessel according to thepresent invention.

FIG. 27 is another front view of the vessel of FIG. 21.

FIG. 28 is a front view of the vessel of FIG. 21 inserted into aheat-exchanging module of the instrument of FIG. 10.

FIG. 29 is an exploded view of a support structure for holding theplates of FIG. 23.

FIGS. 30-31 are assembled views of the support structure of FIG. 29.

FIG. 32 is an isometric view showing the exterior of one the opticsassemblies in the heat-exchanging module of FIG. 28.

FIG. 33 is an isometric view of the plates of FIG. 23 in contact withthe optics assembly of FIG. 32.

FIG. 34 is a partially cut away, isometric view of the reaction vesselof FIG. 21 inserted between the plates of FIG. 23. Only the lowerportion of the vessel is included in the figure.

FIG. 35 is a schematic block diagram of the electronics of theheat-exchanging module of FIG. 28.

FIG. 36 is an isometric view of an apparatus for disrupting cells orviruses according to another embodiment of the invention.

FIG. 37 is a cross sectional view of the apparatus of FIG. 36.

FIG. 38 is an exploded view of a container used in the apparatus of FIG.36.

FIG. 39 is a cross sectional view of the container of FIG. 38.

FIG. 40 is a schematic block diagram of a fluidic system incorporatingthe apparatus of FIG. 36.

FIG. 41 is a cross sectional view of another container for use in theapparatus of FIG. 36. An ultrasonic horn is in contact with a wall ofthe container that curves outwardly towards the horn.

FIG. 42 is a cross-sectional view of the wall of FIG. 41.

FIGS. 43A-43B are isometric views of opposite sides of another wallsuitable for use in a container for holding cells or viruses to bedisrupted.

FIG. 44 is a partially cut-away, isometric view of a containerincorporating the wall of FIGS. 43A-43B.

FIG. 45 is a bottom plan view of the container of FIG. 44.

FIG. 46 is a partially exploded, isometric view of a container forholding cells or viruses to be disrupted according to another embodimentof the invention.

FIG. 47 is a front view of the container of FIG. 46.

FIG. 48 is another schematic, front view of the container of FIG. 46.

FIG. 49 is a side view of the container of FIG. 46.

FIG. 50 is a view of a pipette inserted into the container of FIG. 46.The container is holding beads for rupturing cells or viruses.

FIG. 51 is an isometric view of the container of FIG. 46 inserted intoan apparatus for disrupting cells or viruses.

FIG. 52 is a different isometric view of the container of FIG. 46inserted into the apparatus of FIG. 51.

FIG. 53 is a partially cut-away, isometric view of the apparatus of FIG.51.

FIG. 54 is an isometric view of a holder for holding the container ofFIG. 46.

FIG. 55 is another isometric view of the apparatus of FIG. 51 in whichseveral parts of the apparatus have been removed to show an ultrasonichorn contacting the container of FIG. 46.

FIG. 56 is a schematic side view of the container of FIG. 46 insertedinto the apparatus of FIG. 51 for disruption of the cells or virusescontained in the container.

DETAILED DESCRIPTION

The present invention provides an apparatus and method for analyzing afluid sample. In a first embodiment, the invention provides a cartridgefor separating a desired analyte from a fluid sample and for holding theanalyte for a chemical reaction. The fluid sample may be a solution orsuspension. In a particular use, the sample may be a bodily fluid (e.g.,blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, orother bodily fluids). Alternatively, the sample may be a solid madesoluble or suspended in a liquid or the sample may be an environmentalsample such as ground or waste water, soil extracts, pesticide residues,or airborne spores placed in a fluid. Further, the sample may be mixedwith one or more chemicals, reagents, diluents, or buffers. The samplemay be pretreated, for example, mixed with chemicals, centrifuged,pelleted, etc., or the sample may be in a raw form.

The desired analyte is typically intracellular material (e.g., nucleicacid, proteins, carbohydrates, lipids, bacteria, or intracellularparasites). In a preferred use, the analyte is nucleic acid which thecartridge separates from the fluid sample and holds for amplification(e.g., using PCR) and optical detection. As used herein, the term“nucleic acid” refers to any synthetic or naturally occurring nucleicacid, such as DNA or RNA, in any possible configuration, i.e., in theform of double-stranded nucleic acid, single-stranded nucleic acid, orany combination thereof.

FIG. 1 shows an isometric view of a cartridge 20 according to thepreferred embodiment. The cartridge 20 is designed to separate nucleicacid from a fluid sample and to hold the nucleic acid for amplificationand detection. The cartridge 20 has a body comprising a top piece 22, amiddle piece 24, and a bottom piece 26. An inlet port for introducing afluid sample into the cartridge is formed in the top piece 22 and sealedby a cap 30. Six pressure ports 32 are also formed in the top piece 22.The pressure ports 32 are for receiving nozzles from pressure sources,e.g., pumps or vacuums. The cartridge also includes alignment legs 28extending from the bottom piece 26 for positioning the cartridge 20 inan instrument (described below with reference to FIG. 10). Indentationsor depressions 38A, 38B, and 38C are formed in the top and middle pieces22, 24. The indentations are for receiving optical sensors that detectfluid flow in the cartridge 20. The cartridge 20 further includes vents34, 36. Each pressure port and vent preferably includes a hydrophobicmembrane that allows the passage of gas but not liquid into or out ofthe vents and pressure ports. Modified acrylic copolymer membranes arecommercially available from, e.g., Gelman Sciences (Ann Arbor, Mich.)and particle-track etched polycarbonate membranes are available fromPoretics, Inc. (Livermore, Calif.).

FIG. 2 is an isometric view showing the underside of the cartridge 20.Nine holes 60 are formed in the bottom piece 26 for receiving valveactuators that open and close valves in the cartridge 20. A hole 62 isalso formed in the bottom piece 26 for receiving a transducer (describedin detail below with reference to FIG. 5). The cartridge 20 alsoincludes a reaction vessel 40 extending outwardly from the body of thecartridge. The vessel 40 has a reaction chamber 42 for holding areaction mixture (e.g., nucleic acid mixed with amplification reagentsand fluorescent probes) for chemical reaction and optical detection. Oneof the flow paths in the cartridge carries the reaction mixture to thechamber 42 for chemical reaction and optical detection. The vessel 40extends outwardly from the body of the cartridge 20 so that the vessel40 may be inserted between a pair of opposing thermal plates (forheating and cooling the chamber 42) without the need for decoupling thevessel 40 from the rest of the cartridge 20. This greatly reduces therisk of contamination and/or spilling. The vessel 40 may be integrallyformed with the body of the cartridge (e.g., integrally molded withmiddle piece 24). It is presently preferred, however, to produce thevessel 40 as a separate element that is coupled to the body duringmanufacture of the cartridge.

FIGS. 3-4 show exploded views of the cartridge. As shown in FIG. 3, themiddle piece 24 has multiple chambers formed therein. In particular, themiddle piece 24 includes a sample chamber 65 for holding a fluid sampleintroduced through the inlet port 64, a wash chamber 66 for holding awash solution, a reagent chamber 67 for holding a lysing reagent, awaste chamber 68 for receiving used sample and wash solution, aneutralizer chamber 70 for holding a neutralizer, and a master mixchamber 71 for holding a master mix (e.g., amplification reagents andfluorescent probes) and for mixing the reagents and probes with analyteseparated from the fluid sample. The sample chamber 65 optionallyincludes a side compartment 155 having slightly lower walls than thesample chamber 65. The side compartment 155 is for visually indicatingto a user when sufficient sample has been added to the sample chamber65, i.e., when the liquid level in the chamber 65 is high enough tospill over into the compartment 155.

The top piece 22 includes the vents 34, 36 and the six pressure ports32, as previously described. An elastomeric membrane or gasket 61 ispositioned and squeezed between the pieces 22, 24 to seal the variouschannels and chambers formed in the pieces. The middle piece 24preferably includes multiple sealing lips to ensure that the gasket 61forms an adequate seal. In particular, the middle piece 24 preferablyincludes sealing lips 73 surrounding each of the chambers 65, 66, 67,68. 70, and 71. The middle piece 24 also includes support walls 75around the perimeter, and intermediate sealing lips 76. The sealing lips73, 76 and support walls 75 locally compress the gasket 61 and achieve aseal.

As shown in FIG. 4, the middle piece 24 has formed in its undersidevarious channels, one of which leads to a lysing chamber 86. The chamber86 is aligned with the hole 62 in the bottom piece 26 so that atransducer (e.g., an ultrasonic horn) may be inserted through the hole62 to generate dynamic pressure pulses or pressure waves in the lysingchamber 86. The middle piece 24 also has nine valve seats 84 formed inits bottom surface. The valve seats 84 are aligned with the nine holes60 in the bottom piece 26 so that valve actuators may be insertedthrough the holes 60 into the valve seats 84.

An elastomeric membrane or gasket 61 is positioned and squeezed betweenthe pieces 24, 26 to seal the various channels, valve seats, and chamberformed in the middle piece 24. The middle piece 24 preferably includesmultiple sealing lips to ensure that the gasket 63 forms an adequateseal. In particular, the middle piece 24 preferably includes sealinglips 73 surrounding the lysing chamber 86, valve seats 84, and variouschannels. The middle piece 24 also includes support walls 75 around itsperimeter, and intermediate sealing lips 76. The sealing lips 73, 76 andsupport walls 75 locally compress the gasket 63 and achieve a seal. Inaddition to sealing various channels and chambers, the gasket 63 alsofunctions as a valve stem by compressing, when actuated through one ofthe holes 60, into a corresponding valve seat 84, thus shutting one ofthe flow channels in the middle piece 24. This valve action is discussedin greater detail below with reference to FIGS. 15-16.

The gasket 63 also forms the bottom wall of the lysing chamber 86against which a transducer is placed to effect disruption of cells orviruses in the chamber 86. Each of the gaskets 61, 63 is preferablycomposed of an elastomer. Suitable gasket materials are silicone rubber,neoprene, EPDM, or any other compliant material. Each of the gaskets 61,63 preferably has a thickness in the range of 0.005 to 0.125 inches(0.125 to 3.175 mm), and more preferably in the range of 0.01 to 0.06inches (0.25 to 1.5 mm), with a presently preferred thickness of 0.031inches (0.79 mm). The thickness is selected to ensure that the gasket issufficiently compliant to seal the channels and chambers, to compressinto the valve seats 84 when forced, and to expand under pressure tocontact the transducer.

As shown in FIG. 3, the middle piece 24 includes a slot 79 through whichthe reaction vessel 40 is inserted during assembly of the cartridge. Thevessel 40 has two fluid ports 41, 43 for adding and removing fluid fromthe vessel. When the top piece 22 is sealed to the middle piece 24 viathe gasket 61, the ports 41, 43 are placed into fluidic communicationwith channels 80, 81, respectively, that are formed in the top piece 22(see FIG. 4). The gasket 61 seals the respective fluidic interfacesbetween the ports 41, 43 and the channels 80, 81. The top, middle, andbottom pieces 22, 24, 26 are preferably injection molded parts made of apolymeric material such as polypropylene, polycarbonate, or acrylic.Although molding is preferred for mass production, it also possible tomachine the top, middle, and bottom pieces 22, 24, 26. The pieces 22,24, 26 may be held together by screws or fasteners. Alternatively,ultrasonic bonding, solvent bonding, or snap fit designs could be usedto assemble the cartridge.

FIG. 4 also shows a filter ring 88. The filter ring 88 compresses andholds a stack of filters in the lysing chamber 86. FIG. 6 shows anexploded view of a filter stack 87. The purpose of the filter stack 87is to capture cells or viruses from a fluid sample as the sample flowsthrough the lysing chamber 86. The captured cells or viruses are thendisrupted (lysed) in the chamber 86. The cells may be animal or plantcells, spores, bacteria, or microorganisms. The viruses may be any typeof infective agents having a protein coat surrounding an RNA or DNAcore.

The filter stack 87 comprises a gasket 93, a first filter 94, a gasket95, a second filter 97 having a smaller pore size than the first filter94, a gasket 98, a third filter 100 having a smaller pore size than thesecond filter 97, a gasket 101, a woven mesh 102, and a gasket 103. Thefilter stack also preferably includes a first set of beads 96 disposedbetween the first and second filters 94 and 97 and a second set of beads99 disposed between the second and third filters 97 and 100. The filterring 88 compresses the filter stack 87 into the lysing chamber 86 sothat the gasket 93 is pressed against the filter 94, the filter 94 ispressed against the gasket 95, the gasket 95 is pressed against thefilter 97, the filter 97 is pressed against the gasket 98, the gasket 98is pressed against the filter 100, the filter 100 is pressed against thegasket 101, the gasket 101 is pressed against the mesh 102, the mesh 102is pressed against the gasket 103, and the gasket 103 is pressed againstthe outer perimeter of the bottom wall of the lysing chamber 86. Thegasket 95 is thicker than the average diameter of the beads 96 so thatthe beads are free to move in the space between the filters 94 and 97.Similarly, the gasket 98 is thicker than the average diameter of thebeads 99 so that the beads 99 are free to move in the space between thefilters 97 and 100. A fluid sample flowing through the channel 106 intothe lysing chamber 86 first flows through filter 94, then through filter97, next through filter 100, and lastly through the mesh 102. Afterflowing through the filter stack 87, the sample flows along flow ribs 91formed in the top of the lysing chamber 86 and through an outlet channel(not shown in FIG. 6).

Referring to FIG. 5, the cells or viruses captured in the filter stack(not shown in FIG. 5 for illustrative clarity) are lysed by coupling atransducer 92 (e.g., an ultrasonic horn) directly to the wall of thelysing chamber 86. In this embodiment, the wall of the lysing chamber 86is formed by the flexible gasket 63. The transducer 92 should directlycontact an external surface of the wall. The term “external surface” isintended to mean a surface of the wall that is external to the lysingchamber 86. The transducer 92 is a vibrating or oscillating device thatis activated to generate dynamic pressure pulses or pressure waves inthe chamber 86. The pressure waves agitate the beads 96, 99 (FIG. 6),and the movement of the beads ruptures the captured cells or viruses. Ingeneral, the transducer for contacting the wall of the lysing chamber 86may be an ultrasonic, piezoelectric, magnetostrictive, or electrostatictransducer. The transducer may also be an electromagnetic device havinga wound coil, such as a voice coil motor or a solenoid device. It ispresently preferred that the actuator be an ultrasonic transducer, suchas an ultrasonic horn. Suitable horns are commercially available fromSonics & Materials, Inc. having an office at 53 Church Hill, Newton,Conn. 06470-1614 USA. Alternatively, the ultrasonic transducer maycomprise a piezoelectric disk or any other type of ultrasonic transducerthat may be coupled to the container. It is presently preferred to usean ultrasonic horn because the horn structure is highly resonant andprovides for repeatable and sharp frequency of excitation and largemotion of the horn tip.

As previously described in FIG. 6, the filter stack includes a gasket atboth of its ends. As shown in FIG. 5, the middle cartridge piece 24 hasa sealing lip 90 against which the gasket at one end of the filter stackis compressed. The gasket at the other end of the filter stack iscompressed by the filter ring 88 to form a seal. The gasket material mayexpand into the relief area outside of the sealing lip 90. The width ofthe sealing lip 90 is small (typically 0.5 mm) so that an excessiveamount of force is not required to achieve a sufficient seal.

The filter ring 88 is held between the filter stack and the cartridgegasket 63. The cartridge gasket 63 is held between the middle piece 24and the bottom piece 26 by a sealing lip 406. Force is thereforetransferred from the bottom piece 26 through the gasket 63 to the filterring 88 and finally to the filter stack. The filter ring 88 contains acontact lip 404 that contacts the gasket 63. The contact lip 404 is nota primary sealing lip (though it will seal) but a force transfermechanism. The width of the contact lip 404 is larger than the width ofthe sealing lip 90 to ensure that deformation and sealing action occursin the filter stack and not taken up in squeezing the cartridge gasket63. The cartridge middle piece 24 also has a sealing lip 406 thatsurrounds the filter ring 88. This is an active sealing area that shouldnot be compromised by the presence of the filter ring 88. For thisreason, there is a gap 407 between the sealing lip 406 and the contactlip 404 on the filter ring 88. The gap 407 is provided to allow thegasket 63 to extrude into the gap 407 as it is compressed by the sealinglip 406 and the contact lip 404. If the contact lip 404 comes to adifferent elevation than the sealing lip 406, the seal will not becompromised because of the gap 407 and the distance between the lips 404and 406.

Referring again to FIG. 6, the filter stack 87 is effective forcapturing cells or viruses as a fluid sample flows through the stack 87without clogging of any of the filters 94, 97, 100 in the stack. Thefirst filter 94 (having the largest pore size) filters out coarsematerial such as salt crystals, cellular debris, hair, tissue, etc. Thesecond filter 97 (having the medium pore size) captures cells or virusesin the fluid sample. The third filter 100 (having the smallest poresize) captures smaller cells or viruses in the sample. The filter stack87 thus enables the simultaneous capture of differently sized samplecomponents without clogging of the filters. The average pore size of thefirst filter 94 is selected to be small enough to filter coarse materialfrom the fluid sample (e.g., salt crystals, cellular debris, hair,tissue) yet large enough to allow the passage of the target cells orviruses containing the desired analyte (e.g., nucleic acid or proteins).In general, the pore size of the first filter 94 should be in the rangeof about 2 to 25 μm, with a presently preferred pore size of about 5 μm.

The average pore sizes of the second and third filters are selected independence upon the average size of the target cells or viruses thatcontain the desired analyte(s). For example, in one embodiment, thefilter stack 87 is used to capture gonorrhea (GC) and chlamydia (Ct)organisms to determine the presence of the diseases in the fluid sample.The GC and Ct organisms have different average diameters, about 1 to 2μm for GC organisms and about 0.3 μm for Ct organisms. In thisembodiment, the second filter 97 has an average pore size of about 1.2μm while the third filter 100 has an average pore size of about 0.22 μmso that most of the GC organisms are captured by the second filter 97while most of the Ct organisms are captured by the third filter 100. Thefilter stack thus enables the simultaneous capture of differently sizedtarget organisms and does so without clogging of the filters. The poresizes of the filters 97, 100 may be selected to capture desired cells orviruses of any size, and the scope of the invention is not limited tothe specific example given.

The filter stack 87 is also useful for disrupting the captured cells orviruses to release the intracellular material (e.g., nucleic acid)therefrom. The first and second sets of beads 96, 99 serve two usefulpurposes in this regard. First, the beads are agitated by dynamicpressure pulses or pressure waves generated by the transducer. Themovement of the beads ruptures the captured cells or viruses. Second,the beads may shear the nucleic acid released from the lysed cells orviruses so that the strands of nucleic acid are sufficiently short toflow through the filters and out of the lysing chamber 86. Suitablebeads for rupturing cells or viruses include borosilicate glass, limeglass, silica, and polystyrene beads.

The beads may be porous or non-porous and preferably have an averagediameter in the range of 1 to 200 μm. The average diameter of the beads96, 99 is selected in dependence upon the intended target cells orviruses to be ruptured by the beads. The average diameter of the beads96 in the first set may be equal to the average diameter of the beads 99in the second set. Alternatively, when the first set of beads 96 is usedto rupture a type of target cell or virus that differs from the type ofcell or virus to be ruptured by the second set of beads 99, it isadvantageous to select the average diameter of the beads such that theaverage diameter of the beads 96 in the first set differs from theaverage diameter of the beads 99 in the second set. For example, whenthe filter stack is used to capture GC and Ct cells as described above,the beads 96 are 20 μm diameter borosilicate glass beads for rupturingthe GC organisms and the beads 99 are 106 μm diameter soda lime glassbeads for rupturing the Ct organisms. Each of the silicone gaskets 95,98 should be sufficiently thick to allow room for the beads 96, 99 tomove and rupture the cells or viruses.

The mesh 102 also serves two useful purposes. First the mesh providessupport to the filter stack 87. Second, the mesh breaks up air bubblesso that the bubbles can be channeled through the flow ribs 91 and out ofthe lysing chamber 86. To effectively break up or reduce the size of theair bubbles, the mesh 102 preferably has a small pore size. Preferably,it is a woven polypropylene mesh having an average pore size of about 25μm. To ensure that the air bubbles can escape from the lysing chamber86, it is desirable to use the cartridge in an orientation in whichliquid flows up (relative to gravity) through the filter stack 87 andthe lysing chamber 86. The upward flow through the chamber 86 aids theflow of air bubbles out of the chamber 86. Thus, the inlet port forentry of fluids into the chamber 86 should generally be at the lowestpoint in the chamber, while the exit port should be at the highest.

Many different embodiments of the filter stack are possible. Forexample, in one alternative embodiment, the filter stack has only twofilters and one set of beads disposed between the filters. The firstfilter has the largest pore size (e.g., 5 μm) and filters out coarsematerial such as salt crystals, cellular debris, hair, tissue, etc. Thesecond filter has a pore size smaller than the first filter and slightlysmaller than the target cells or viruses to be captured. Such a filterstack is described below with reference to FIG. 38. In anotherembodiment of the cartridge, the filter having the largest pore size(for filtering the coarse material) is positioned in a filter chamber(not shown) that is positioned upstream of the lysing chamber 86. Achannel connects to the filter chamber to the lysing chamber 86. In thisembodiment, a fluid sample flows first through the coarse filter in thefilter chamber and then through a second filter in the lysing chamber totrap the target cells or viruses in the lysing chamber.

Further, the beads in the filter stack may have a binding affinity fortarget cells or viruses in the fluid sample to facilitate capture of thetarget cells or viruses. For example, antibodies or certain receptorsmay be coated onto the surface of the beads to bind target cells in thesample. Moreover, the lysing chamber 86 may contain two different typesof beads for interacting with target cells or viruses. For example, thelysing chamber may contain a first set of beads coated with antibodiesor receptors for binding target cells or viruses and a second set ofbeads (intermixed with the first set) for rupturing the captured cellsor viruses. The beads in the lysing chamber 86 may also have a bindingaffinity for the intracellular material (e.g., nucleic acid) releasedfrom the ruptured cells or viruses. Such beads are useful for isolatingtarget nucleic acid for subsequent elution and analysis. For example,the lysing chamber may contain silica beads to isolate DNA or cellulosebeads with oligo dT to isolate messenger RNA for RT-PCR. The lysingchamber 86 may also contain beads for removing unwanted material (e.g.,proteins, peptides) or chemicals (e.g., salts, metal ions, ordetergents) from the sample that might inhibit PCR. For example, thechamber 86 may contain ion exchange beads for removing proteins.Alternatively beads having metal ion chelators such as iminodiaceticacid will remove metal ions from biological samples.

FIGS. 21-22 illustrate the reaction vessel 40 in greater detail. FIG. 21shows a partially exploded view of the vessel 40, and FIG. 22 shows afront view of the vessel 40. The vessel 40 includes the reaction chamber42 (diamond-shaped in this embodiment) for holding a reaction mixture.The vessel 40 is designed for optimal heat transfer to and from thereaction mixture and for efficient optical viewing of the mixture. Thethin shape of the vessel contributes to optimal thermal kinetics byproviding large surfaces for thermal conduction and for contactingthermal plates. In addition, the walls of the vessel provide opticalwindows into the chamber 42 so that the entire reaction mixture can beoptically interrogated. In more detail to FIGS. 21-22, the reactionvessel 40 includes a rigid frame 46 that defines the side walls 57A,57B, 59A, 59B of the reaction chamber 42. The frame 46 also defines aninlet port 41 and a channel 50 connecting the port 41 to the chamber 42.The frame 46 also defines an outlet port 43 and a channel 52 connectingthe port 43 to the chamber 42. The inlet port 41 and channel 50 are usedto add fluid to the chamber 42, and the channel 52 and outlet port 43are used for exit of fluid from the chamber 42. Alignment prongs 44A,44B are used to position the vessel 40 correctly during assembly of thecartridge.

As shown in FIG. 21, the vessel 40 also includes thin, flexible sheetsattached to opposite sides of the rigid frame 46 to form opposing majorwalls 48 of the chamber. (The major walls 48 are shown in FIG. 1exploded from the rigid frame 46 for illustrative clarity). The reactionchamber 42 is thus defined by the rigid side walls 57A, 57B, 59A, 59B ofthe frame 46 and by the opposing major walls 48. The opposing majorwalls 48 are sealed to opposite sides of the frame 46 such that the sidewalls 57A, 57B, 59A, 59B connect the major walls 48 to each other. Thewalls 48 facilitate optimal thermal conductance to the reaction mixturecontained in the chamber 42. Each of the walls 48 is sufficientlyflexible to contact and conform to a respective thermal surface, thusproviding for optimal thermal contact and heat transfer between thethermal surface and the reaction mixture contained in the chamber 42.Furthermore, the flexible walls 48 continue to conform to the thermalsurfaces if the shape of the surfaces changes due to thermal expansionor contraction during the course of the heat-exchanging operation.

As shown in FIG. 23, the thermal surfaces for contacting the flexiblewalls 48 are preferably formed by a pair of opposing plates 190A, 190Bpositioned to receive the chamber 42 between them. When the chamber 42of the vessel 40 is inserted between the plates 190A, 190B, the innersurfaces of the plates contact the walls 48 and the flexible wallsconform to the surfaces of the plates. The plates are preferably spaceda distance from each other equal to the thickness T of the chamber 42 asdefined by the thickness of the frame 46. In this position, minimal orno gaps are found between the plate surfaces and the walls 48. Theplates may be heated and cooled by various thermal elements to inducetemperature changes within the chamber 42, as is described in greaterdetail below.

The walls 48 are preferably flexible films of polymeric material such aspolypropylene, polyethylene, polyester, or other polymers. The films mayeither be layered, e.g., laminates, or the films may be homogeneous.Layered films are preferred because they generally have better strengthand structural integrity than homogeneous films. In particular, layeredpolypropylene films are presently preferred because polypropylene is notinhibitory to PCR. Alternatively, the walls 48 may comprise any othermaterial that may be formed into a thin, flexible sheet and that permitsrapid heat transfer. For good thermal conductance, the thickness of eachwall 48 is preferably between about 0.003 to 0.5 mm, more preferablybetween 0.01 to 0.15 mm, and most preferably between 0.025 to 0.08 mm.

Referring again to FIG. 22, the vessel 40 also preferably includesoptical windows for in situ optical interrogation of the reactionmixture in the chamber 42. In the preferred embodiment, the opticalwindows are the side walls 57A, 57B of the rigid frame 46. The sidewalls 57A, 57B are optically transmissive to permit excitation of thereaction mixture in the chamber 42 through the side wall 57A anddetection of light emitted from the chamber 42 through the side wall57B. Arrows A represent illumination beams entering the chamber 42through the side wall 57A and arrows B represent emitted light (e.g.,fluorescent emission from labeled analytes in the reaction mixture)exiting the chamber 42 through the side wall 57B.

The side walls 57A, 57B are preferably angularly offset from each other.It is usually preferred that the walls 57A, 57B are offset from eachother by an angle of about 90°. A 90° angle between excitation anddetection paths assures that a minimum amount of excitation radiationentering through the wall 57A will exit through wall 57B. In addition,the 90° angle permits a maximum amount of emitted light (e.g.fluorescence) to be collected through wall 57B. The walls 57A, 57B arepreferably joined to each other to form a “V” shaped intersection at thebottom of the chamber 42. Alternatively, the angled walls 57A, 57B neednot be directly joined to each other, but may be separated by anintermediary portion, such as another wall or various mechanical orfluidic features which do not interfere with the thermal and opticalperformance of the vessel. For example, the walls 57A, 57B may meet at aport which leads to another processing area in communication with thechamber 42, such as an integrated capillary electrophoresis area. In thepresently preferred embodiment, a locating tab 58 extends from the frame46 below the intersection of walls 57A, 57B. The tab 58 is used toproperly position the vessel 40 in a heat-exchanging module describedbelow with reference to FIG. 28.

Optimum optical sensitivity may be attained by maximizing the opticalpath length of the light beams exciting the labeled analyte in thereaction mixture and the emitted light that is detected, as representedby the equation:I _(o) /I _(i) =C*L*A,where I_(o) is the illumination output of the emitted light in volts,photons or the like, C is the concentration of analyte to be detected,I_(i) is the input illumination, L is the path length, and A is theintrinsic absorptivity of the dye used to label the analyte.

The thin, flat reaction vessel 40 of the present invention optimizesdetection sensitivity by providing maximum optical path length per unitanalyte volume. Referring to FIGS. 23 and 27, the vessel 40 ispreferably constructed such that each of the sides walls 57A, 57B, 59A,59B of the chamber 42 has a length L in the range of 1 to 15 mm, thechamber has a width W in the range of 1.4 to 20 mm, the chamber has athickness T in the range of 0.5 to 5 mm, and the ratio of the width W ofthe chamber to the thickness T of the chamber is at least 2:1. Theseparameters are presently preferred to provide a vessel having arelatively large average optical path length through the chamber, i.e. 1to 15 mm on average, while still keeping the chamber sufficiently thinto allow for extremely rapid heating and cooling of the reaction mixturecontained therein. The average optical path length of the chamber 42 isthe distance from the center of the side wall 57A to the center of thechamber 42 plus the distance from the center of the chamber 42 to thecenter of the side wall 57B.

More preferably, the vessel 40 is constructed such that each of thesides walls 57A, 57B, 59A, 59B of the chamber 42 has a length L in therange of 5 to 12 mm, the chamber has a width W in the range of 7 to 17mm, the chamber has a thickness T in the range of 0.5 to 2 mm, and theratio of the width W of the chamber to the thickness T of the chamber isat least 4:1. These ranges are more preferable because they provide avessel having both a larger average optical path length (i.e., 5 to 12mm) and a volume capacity in the range of 12 to 100 μl while stillmaintaining a chamber sufficiently thin to permit extremely rapidheating and cooling of a reaction mixture. The relatively large volumecapacity provides for increased sensitivity in the detection of lowconcentration analytes, such as nucleic acids.

In the preferred embodiment, the reaction vessel 40 has a diamond-shapedchamber 42 defined by the side walls 57A, 57B, 59A, 59B, each of theside walls has a length of about 10 mm, the chamber has a width of about14 mm, the chamber has a thickness T of 1 mm as defined by the thicknessof the frame 46, and the chamber has a volume capacity of about 100 μl.This reaction vessel provides a relatively large average optical pathlength of 10 mm through the chamber 42. Additionally, the thin chamberallows for extremely rapid heating and/or cooling of the reactionmixture contained therein. The diamond-shape of the chamber 42 helpsprevent air bubbles from forming in the chamber as it is filled with thereaction mixture and also aids in optical interrogation of the mixture.

Referring again to FIG. 22, the frame 46 is preferably made of anoptically transmissive material, e.g., a polycarbonate or clarifiedpolypropylene, so that the side walls 57A, 57B are opticallytransmissive. As used herein, the term optically transmissive means thatone or more wavelengths of light may be transmitted through the walls.In the preferred embodiment, the optically transmissive walls 57A, 57Bare substantially transparent. In addition, one or more optical elementsmay be present on the optically transmissive side walls 57A, 57B. Theoptical elements may be designed, for example, to maximize the totalvolume of solution which is illuminated by a light source, to focusexcitation light on a specific region of the chamber 42, or to collectas much fluorescence signal from as large a fraction of the chambervolume as possible. In alternative embodiments, the optical elements maycomprise gratings for selecting specific wavelengths, filters forallowing only certain wavelengths to pass, or colored lenses to providefiltering functions. The wall surfaces may be coated or comprisematerials such as liquid crystal for augmenting the absorption ofcertain wavelengths. In the presently preferred embodiment, theoptically transmissive walls 57A, 57B are substantially clear, flatwindows having a thickness of about 1 mm.

The side walls 59A, 59B preferably includes reflective faces 56 whichinternally reflect light trying to exit the chamber 42 through the sidewalls 59A, 59B. The reflective faces 56 are arranged such that adjacentfaces are angularly offset from each other by about 90°. In addition,the frame 46 defines open spaces between the side walls 59A, 59B and thesupport ribs 53. The open spaces are occupied by ambient air that has adifferent refractive index than the material composing the frame (e.g.,plastic). Due to the difference in the refractive indexes, thereflective faces 56 are effective for internally reflecting light tryingto exit the chamber through the walls 59A, 59B and provide for increaseddetection of optical signal through the walls 57A, 57B. Preferably, theoptically transmissive side walls 57A, 57B define the bottom portion ofthe diamond-shaped chamber 42, and the retro-reflective side walls 59A,59B define the top portion of the chamber.

A preferred method for fabricating the reaction vessel 40 will now bedescribed with reference to FIGS. 21-22. The reaction vessel 40 may befabricated by first molding the rigid frame 46 using known injectionmolding techniques. The frame 46 is preferably molded as a single pieceof polymeric material, e.g., clarified polypropylene. After the frame 46is produced, thin, flexible sheets are cut to size and sealed toopposite sides of the frame 46 to form the major walls 48 of the chamber42. The major walls 48 are preferably cast or extruded films ofpolymeric material, e.g., polypropylene films, that are cut to size andattached to the frame 46 using the following procedure. A first piece offilm is placed over one side of the frame 46. The frame 46 preferablyincludes a tack bar 47 for aligning the top edge of the film. The filmis placed over the bottom portion of the frame 46 such that the top edgeof the film is aligned with the tack bar 47 and such that the filmcompletely covers the bottom portion of the frame 46 below the tack bar47. The film should be larger than the bottom portion of the frame 46 sothat it may be easily held and stretched flat across the frame. The filmis then cut to size to match the outline of the frame by clamping to theframe the portion of the film that covers the frame and cutting away theportions of the film that extend past the perimeter of the frame using,e.g., a laser or die. The film is then tack welded to the frame,preferably using a laser.

The film is then sealed to the frame 46, preferably by heat sealing.Heat sealing is presently preferred because it produces a strong sealwithout introducing potential contaminants to the vessel as the use ofadhesive or solvent bonding techniques might do. Heat sealing is alsosimple and inexpensive. The heat sealing may be performed using, e.g., aheated platen. An identical procedure may be used to cut and seal asecond sheet to the opposite side of the frame 46 to complete thechamber 42. Many variations to this fabrication procedure are possible.For example, in an alternative embodiment, the film is stretched acrossthe bottom portion of the frame 46 and then sealed to the frame prior tocutting the film to size. After sealing the film to the frame, theportions of the film that extend past the perimeter of the frame are cutaway using, e.g., a laser or die.

Although it is presently preferred to mold the frame 46 as a singlepiece, it is also possible to fabricate the frame from multiple pieces.For example, the side walls 57A, 57B forming the angled optical windowsmay be molded from polycarbonate, which has good optical transparency,while the rest of the frame is molded from polypropylene, which isinexpensive and compatible with PCR. The separate pieces can be attachedtogether in a secondary step. For example, the side walls 57A, 57B maybe press-fitted and/or bonded to the remaining portion of the frame 46.The flexible walls 48 may then be attached to opposite sides of theframe 46 as previously described.

Referring again to FIG. 3, it is presently preferred to use a gasket 61to seal the ports 41, 43 of the vessel 40 to corresponding channels 80,81 (FIG. 4) in the cartridge body. Alternatively, fluidic seals may beestablished using a luer fitting, compression fitting, or swagedfitting. In another embodiment, the cartridge body and frame of thevessel 40 are molded as a single part, and the flexible major walls ofthe vessel are heat-sealed to opposite sides of the frame.

Referring again to FIG. 22, the chamber 42 is filled by forcing liquid(e.g., a reaction mixture) to flow through the port 41 and the channel50 into the chamber 42. The liquid may be forced to flow into thechamber 42 using differential pressure (i.e., either pushing the liquidthrough the inlet port 41 or aspirating the liquid by applying a vacuumto the outlet port 43). As the liquid fills the chamber 42, it displacesair in the chamber. The displaced air exits the chamber 42 through thechannel 52 and the port 43. For optimal detection of analyte in thechamber 42, the chamber should not contain air bubbles. To help preventthe trapping of air bubbles in the chamber 42, the connection betweenthe chamber 42 and the outlet channel 52 should be at the highest point(with respect to gravity) in the chamber 42. This allows air bubbles inthe chamber 42 to escape without being trapped. Thus, the vessel 40 isdesigned to be used in the vertical orientation shown in FIG. 22.

FIG. 25 shows another vessel 206 designed to be used in a horizontalorientation. The vessel 206 has an inlet port 41 and an inlet channel 50connecting the inlet port 41 to the bottom of the chamber 42. The vesselalso has an outlet port 43 and an outlet channel 50 connecting theoutlet port 43 to the top of the chamber 42. Thus, any air bubbles inthe chamber 42 may escape through the outlet channel 52 without becomingtrapped. FIG. 26 shows another vessel 207 having two inlet ports 41, 45and one outlet port 43. Inlet channels 50, 54 connect the respectiveinlet ports 41, 45 to the chamber 42, and outlet channel 52 connects thechamber 42 to outlet port 43. Many other different embodiments of thevessel are also possible. In each embodiment, it is desirable toevacuate the chamber 42 from the highest point (with respect to gravity)in the chamber and to introduce liquid into the chamber from a lowerpoint.

FIGS. 15A-15B illustrate two types of valves used in the cartridge. Asshown in FIG. 15A, there are two types of fundamental concepts to thevalve action, and hence two types of valves. The first valve uses acone-shaped or conical valve seat 160 formed in the middle cartridgepiece 24. The valve seat 160 is a depression, recess, or cavity moldedor machined in the middle piece 24. The valve seat 160 is in fluidcommunication with a chamber 167 through a port or channel 157 thatintersects the center of the conical valve seat 160. As shown in FIG.15B, a valve actuator 164 having a spherical surface is forced againstthe elastic membrane 63 and into the valve seat 160, establishing acircular ring of contact between the membrane 63 and the valve seat 160.The kinematic principle is that of a ball seated into a cone. Thecircular seal formed by the membrane 63 and valve seat 160 prevents flowbetween the channel 157 (and hence the chamber 167) and a side channel158 extending from a side of the valve seat 160. The side channel 158 isdefined by the membrane 63 and the middle cartridge piece 24.

As shown in FIG. 15A, the other type of valve controls the cross flowbetween the channel 158 and another side channel 159 formed between themembrane 63 and the middle cartridge piece 24. In this case, a circularring of contact would be ineffective. Instead, the second valvecomprises a recess depression or cavity 161 formed in the middlecartridge piece 24. The cavity 161 separates the channels 158, 159 fromeach other. An end of the channel 158 is positioned on one side of thecavity 161, and an end of the channel 159 is positioned on the oppositeside of the cavity 161. The cavity 161 is defined by a first curvedsurface 162A positioned adjacent the end of the channel 158, a secondcurved surface 162B positioned adjacent the end of the channel 159, anda third surface 163 between the first and second curved surfaces 162A,162B. As shown in FIG. 15B, the curved surfaces provide two valve seatsthat are the primary contact area for the membrane 63 to seal off theflow between the channels 158 and 159. The kinematic principle is thatof a ball (or spherical end on a valve actuator) held by three contactpoints, the upward force on the actuator and the two valve seats 162A,162B.

As shown in FIG. 16A, the first and second curved surfaces 162A, 162Bare preferably concentric spherical surfaces. The valve actuator 164 hasalso has a spherical surface for pressing the membrane 63 tightlyagainst the surfaces 162A, 162B. In addition, each of the surfaces 162A,162B preferably has a spherical radius of curvature R1 equal to thecombined radius of curvature R2 of the valve actuator 164 plus thethickness T of the membrane 63. For example, if the radius of curvatureR2 of the surface of the valve actuator 164 is 0.094 inches and themembrane 63 has a thickness T of 0.031 inches, then the radius ofcurvature R1 of each of the surfaces 162A, 162B is 0.125 inches. Ingeneral, the size and radius of curvature of the valve seats isdependent upon the size of the channels in the cartridge. The valves arepreferably made just large enough to effectively seal the channels butno larger so that dead volume in the cartridge is minimized.

As shown in FIG. 16B, the third surface 163 is recessed from the firstand second surfaces 162A, 162B to provide a gap 166 between the membrane63 and the third surface 163 when the membrane 63 is pressed against thefirst and second surfaces 162A, 162B. Stated another way, the surfaces162A, 162B are raised or elevated from the surface 163. The gap 166ensures that the membrane 63 contacts primarily the valve seats 162A,162B rather than the entire surface of the cavity 161 so that maximumpressure is applied to the valve seats 162A and 162B by the membrane 63.This provides a very strong seal with minimal actuator force required.

Referring again to FIG. 15B, in both types of valves the respectivekinematic principle defines the location of the mating parts. In boththe ball-in-cone concept and the ball-against-two-spherical-surfacesconcept, the ball or spherical shaped valve actuator is permitted toseek its own location as it is forced against the valve seat(s). Thereis a deliberate clearance (e.g., 0.01 to 0.03 inches) between the valveactuator and the hole in the bottom cartridge piece 26 in which theactuator 164 travels so that only the valve seat action defines thelocation of the mating pieces.

The valve actuators can be controlled by a variety of mechanisms. FIGS.17-19 illustrate one such mechanism. As shown in FIG. 17, a valveactuator 172 has a spherical surface for pressing the gasket 63 into avalve seat. The actuator 172 also has a flange 177 on its bottomportion. The cartridge includes an elastic body, such as a spring 174,that pushes against a ledge in the lower cartridge piece 26 to bias thevalve actuator against the gasket 63. The spring 174 is sufficientlystrong to close the valve unless a deliberate force is applied to pulldown the actuator 172. The valves in the cartridge may be kept closed inthis manner for shipping and storage before the cartridge is used. Thus,the cartridge may be preloaded during manufacture with the necessaryreagents and wash solutions to analyze a fluid sample without the fluidsleaking out of the cartridge during shipping and storage.

The actuator pull-down mechanism is usually located in an instrumentinto which the cartridge is placed for sample analysis (one suchinstrument is described in detail below with reference to FIG. 10). Themechanism comprises a sliding guide 175 that rotates a hinged pull-downmember 180 having a jaw 181 for receiving the flange 177 of the actuator172. As shown in FIG. 18, the sliding guide 175 rotates the hingedpull-down member 180 until the flange 177 is positioned within the jaw181. As shown in FIG. 19, a solenoid 146 pulls down the member 180 andthus the valve actuator 172 so that the gasket 63 is released from thevalve seat, thus opening the valve and permitting fluid flow between thechannels 170 and 171.

FIG. 20 illustrates the manner in which fluid flow into and out of thesample chamber, wash chamber, neutralizer chamber, and reagent chambersis controlled in the cartridge. Each of these chambers, as illustratedby a chamber 414 in FIG. 20, is covered by a hydrophobic membrane 410that allows the passage of gas but not liquid therethrough. Thehydrophobic membrane 410 is positioned between the chamber 414 and apressure port 32. The pressure port 32 is formed in the upper cartridgepiece 22 and positioned over the chamber 414. The membrane 410 holdsliquids in the chamber 414 during shipping and storage of the cartridge,even if the cartridge is turned upside down. The pressure port 32 issized to receive a pressure nozzle 182 that is connected to a pressuresource (e.g., a vacuum or pneumatic pump) usually located in theexternal instrument. The nozzle 182 includes an o-ring 184 and a flange415. A spring 185 pushes against the flange 415 to force the nozzle 182into the pressure port 32 so that the o-ring 184 establishes a sealaround the port 32. In operation, positive air pressure or a vacuum isapplied to the chamber 414 through the pressure port 32 to force liquidsout of or into, respectively, the chamber 414.

A conical valve seat 160 (previously described with reference to FIG.1A-15B) is formed in the middle cartridge piece 24 below the chamber 414to control the flow of liquid between the chamber 414 and a connectingchannel 411. The valve is opened and closed by a valve actuator 188having a flange 187 and a spring 188 pressing against the flange to holdthe valve closed until a downward force is applied to the actuator 186.The downward force is preferably supplied by a solenoid that pulls downthe actuator 186 to open the valve. The valve actuator 186 and solenoidare preferably located in the instrument.

FIGS. 7-8 show top and bottom plan views, respectively, of thecartridge. FIG. 9 is a schematic block diagram of the cartridge. Asshown in any of FIGS. 7-9, the cartridge includes a sample chamber 65having a port for adding a fluid sample to the cartridge and a sampleflow path extending from the sample chamber 65. The sample flow pathextends from the sample chamber 65 through a valve 107 and into achannel 106. The channel 106 includes a sensor region 136 in which thechannel 106 has a flat bottom enabling easy optical detection of thepresence of liquid in the channel. The sample flow path continues fromthe channel 106 into the lysing chamber 86 and through the filter stack87. The sample flow path also includes a channel 109 for exit of fluidfrom the lysing chamber 86, a channel 110 having a flat-bottomeddetection region 137, a valve 111, and a channel 112 leading to thevented waste chamber 68 through a valve 114.

The cartridge also includes the wash chamber 66 for holding washsolution and the reagent chamber 67 for holding lysing reagent. The washchamber 66 is connected to the lysing chamber 86 through a valve 115,channel 117, and channel 106. The reagent chamber 67 is connected to thelysing chamber 86 through a valve 119, channel 117, and channel 106.Sample components (e.g., cells or viruses in the sample) are captured inthe filter stack 87 and lysed in the chamber 86 to release targetanalyte (e.g., nucleic acid) from the sample components. The cartridgealso includes an analyte flow path extending from the lysing chamber 86for carrying the analyte separated from the fluid sample to the reactionvessel 40 for chemical reaction and optical detection. The analyte flowpath extends from the chamber 86 through the channel 109, channel 110,and valve 111. After passing through the valve 111, the analyte flowpath diverges from the sample flow path. While the sample flow pathextends though channel 112 into the waste chamber 68, the analyte flowpath diverges into the U-shaped channel 122. The analyte flow path thenextends into and out of the neutralizer chamber 70 through a valve 124.The analyte flow path also passes into and out of the master mix chamber71 through a valve 126. From the master mix chamber 71, the analyte flowpath extends along the channel 122, through a valve 127, through channel80, and into the reaction vessel 40 through the port 41.

The reaction vessel 40 includes the port 41 for adding a reactionmixture to the vessel, and the port 43 for exit of fluids (e.g., air orexcess reaction mixture) from the vessel. The cartridge also includeschannel 81 in fluid communication with the port 43. The channel 81includes a flat-bottomed detection region 130 for detecting the presenceof liquid in the channel. The channel 81 connects to a channel 131(channel 131 extends straight down perpendicular to the page in the topplan view of FIG. 7). Channel 131 connects to a channel 132 which inturn connects to a channel 134 through a valve 133 (channel 134 extendsstraight up perpendicular to the page in the top plan view of FIG. 7).The channel 134 leads to the vent 36 which has a hydrophobic membrane topermit the escape of gas but not liquid from the cartridge. Thechannels, vent and valve positioned downstream from the reaction vessel40 are used to pressurize the chamber 42 of the vessel, as is describedin the operation section below.

The cartridge also includes a first pressure port 105 positioned abovethe sample chamber 65, a second pressure port 116 positioned above thewash chamber 66, a third pressure port 118 positioned above the reagentchamber 67, a fourth pressure port 123 positioned above the neutralizerchamber 70, a fifth pressure port 125 positioned above the master mixchamber 71, and a sixth pressure port 128 positioned at the end of theU-shaped channel 122. The cartridge further includes sensor chambers 120and 121 in fluid communication with the waste chamber 68. The sensorchambers 120 and 121 indicate when predetermined volumes of liquid havebeen received in the waste chamber 68, as is described in detail below.

Referring to FIG. 10, the cartridge is preferably used in combinationwith an instrument 140 designed to accept one or more of the cartridges.For clarity of illustration, the instrument 140 shown in FIG. 10 acceptsjust one cartridge. It is to be understood, however, that the instrumentmay be designed to process multiple cartridges simultaneously. Theinstrument 140 includes a cartridge nest 141 into which the cartridge isplaced for processing. The instrument 140 also includes the transducer92 (e.g., an ultrasonic horn) for generating dynamic pressure pulses orpressure waves in the lysing chamber of the cartridge, nine valveactuators 142 for actuating the nine valves in the cartridge, ninecorresponding solenoids 146 for pulling down the valve actuators, andsix pressure nozzles 145 for interfacing with six corresponding pressureports formed in the cartridge. In addition, the instrument includes oris connected to one or more regulated pressure sources for supplyingpressure to the cartridge through the pressure nozzles 145. Suitablepressure sources include syringe pumps, compressed air sources,pneumatic pumps, or connections to external sources of pressure. Theinstrument further includes three slotted optical sensors 143 and threereflective optical sensors 144.

FIG. 13 illustrates the slotted optical sensors 143 positioned to detectliquid in the sensor chambers 120, 121 and in the reagent chamber 67.Each sensor 143 includes a built in LED and photodiode positioned onopposite sides of the sensor. The LED emits a beam that is detected bythe photodiode if the beam is not substantially refracted. Such slottedoptical sensors are commercially available from a number of suppliers.The cartridge is shaped so that the slotted optical sensors fit aroundthe chambers 67, 120, and 121. The operation of each sensor is asfollows. If liquid is not present in the chamber the sensor surrounds,the beam from the LED is substantially refracted by air in the chamberand the curved inner walls of the chamber and only a weak signal, ifany, is detected by the photodiode since air has an index of refractionthat does not closely match that of the plastic cartridge. If there isliquid present in the chamber, however, the beam from the LED does notrefract or is only slightly refracted and produces a much strongersignal detected by the photodiode since the liquid has an index ofrefraction closely matching that of the plastic cartridge. The opticalsensors 143 are therefore useful for determining the presence or absenceof liquid in the chambers 67, 120, and 121.

FIG. 14 shows a cut-away, schematic side view of the sensor chamber 120in fluid communication with the waste chamber 68 and surrounded by theslotted optical sensor 143. The sensor chamber 120 and sensor 143 areused to indicate when a predetermined volume of liquid is present in thewaste chamber 68. The sensor chamber 120 is partially separated from thewaste chamber 68 by a wall 151 having a spillover rim 152. The height ofthe wall is selected so that when the predetermined volume of liquid isreceived in the waste chamber 68, the liquid spills over the spilloverrim 152 and into the sensor chamber 120. The liquid in the sensorchamber 120 is then detected by the sensor 143.

Referring again to FIG. 13, the cartridge may also include a secondsensor chamber 121 in fluid communication with the waste chamber 68. Thesecond sensor chamber 121 is also separated from the waste chamber 68 bya wall 153 having a spillover rim. The wall 153 is taller than the wall152 so that liquid does not spill over the wall 153 until a secondpredetermined volume of fluid in addition to the first predeterminedvolume of fluid has been received in the waste chamber 68. The sensorchambers 120, 121 and the optical sensors 143 are useful for controllingthe operation of the cartridge. The height of the wall 152 is preferablyselected such that when a fixed volume of fluid sample from the samplechamber 65 has flowed through the sample flow path to the waste chamber68, the sample liquid spills over into the sensor chamber 120 and isdetected. The detection in chamber 120 triggers the release of washsolution from the wash chamber 66 which flows through the sample flowpath to the waste chamber 68. When an incremental volume of the washsolution is received in the chamber 68, liquid spills over the wall 153into the sensor chamber 121 and is detected. The detection of liquid inthe chamber 121 then triggers the release of lysing reagent from thechamber 67. The sensor 143 surrounding the chamber 67 may then be usedto indicate when the chamber 67 is empty, triggering the start ofultrasonic lysis. In an alternative embodiment, the cartridge may havetwo waste chambers, one for sample and one for wash, with each wastechamber having a respective sensor chamber connected thereto.

In-line reflective optical sensors 144 are used to determine thepresence or absence of liquid in the flat-bottomed detection regions130, 136, 137, of channels 81, 106, and 110, respectively (FIG. 7). Eachsensor 144 has a built in emitter and detector that is positioned over aflat-bottomed detection region. The emitter emits a beam that isreflected from the cartridge and detected by the detector. The sensordetects a change in signal when as an air/liquid interface passesthrough the detection region. Optionally, dual emitter reflectiveoptical sensors may be used for a more reliable detection operation.Both types of reflective optical sensors are well known in the art andcommercially available.

Referring again to FIG. 10, the instrument 140 also includes aheat-exchanging module 147 having a slot 148 for receiving the reactionvessel of the cartridge. The module 147 is described in detail belowwith reference to FIG. 28. The instrument 140 further includes a latchmechanism 149 for latching a lid 150 over a cartridge. The cartridgenest 141 includes alignment holes 401 for receiving the legs of thecartridge. The alignment holes 401 ensure proper positioning of thecartridge in the nest 141 so that the pressure nozzles 145, transducer92, and valve actuators 142 fit into the corresponding ports in thecartridge and so that the reaction vessel fits into the slot 148. Thetransducer 92 should be positioned in the instrument 140 such that whenthe cartridge is placed in the nest 141, the transducer contacts thebottom wall of the lysing chamber 86, as shown in the cut-away view ofFIG. 5. In addition, the instrument may include a spring or similarmechanism to bias the transducer 92 against the wall of the lysingchamber 86.

The instrument 140 also includes various conventional equipment notshown in FIG. 10 including a main logic board having a microcontrollerfor controlling the operation of the solenoids 146, transducer 92,heat-exchanging module 147, and optical sensors 143, 144. The instrumentalso includes or is connected to a power supply for powering theinstrument and a pneumatic pump for supplying air pressure through thenozzles 145. The instrument 140 is preferably computer-controlled using,e.g., the microcontroller which is programmed to perform the functionsdescribed in the operation section below. Alternatively, the instrumentmay controlled by a separate computer, or controlled by a combination ofa separate computer and an on-board microcontroller.

FIG. 11 shows an isometric view of the cartridge 20 placed in theinstrument 140 for processing. FIG. 11 shows a partial cut-away view ofthe instrument 140 with the lid 150 closed. Referring again to FIG. 11,a memory or microprocessor chip may optionally be incorporated as partof the cartridge 20. This chip preferably contains information such asthe type of cartridge, program information such as specific protocolsfor the processing of the cartridge, tolerances for accept and reject,serial numbers and lot codes for quality tracking, and provision forstoring the results of the processing. Integrated electronic memory onthe cartridge 20 allows for rapid, easy, and error-free set-up of theinstrument 140 for different fluidic processing protocols. When thecartridge 20 is inserted into the instrument 140, the instrument mayelectronically address the memory on the cartridge, and thusautomatically receive the appropriate set of instructions forcontrolling the time-sequence of fluidic operations to be carried outwith the inserted cartridge. The instrument 140 may simply sequentiallyretrieve and execute each step in the cartridge's memory, or downloadits contents so that the user may edit the sequence using, e.g., thecontroller computer.

If suitable memory is included on the cartridge, such as writable memory(e.g., erasable programmable read-only memory (EPROM), electricallyerasable programmable read-only memory (EEPROM), etc., intermediate andfinal results, based on the sample introduced into the cartridge, couldbe written by the instrument into the cartridge's memory for co-locatedstorage with the physical sample after processing. This is particularlyadvantageous in applications where archiving of samples and results isnecessary, such as forensics. In addition, other information can bestored in the memory on the cartridge, in unalterable (or alterable)forms. For example, cartridge serial number, lot manufactureinformation, and related information could be pre-programmed andunalterable. User data, technician identification number, date of test,location of test and instrument serial number could be unalterablywritten into the cartridge. This allows for easy identification of the“chain of custody” in the handling of a specimen. Engineers skilled inthe art of data storage will recognize that other memory means thanelectronic can be used, such as optically-addressed printed regions(e.g., ink-jet or thermal), magnetic strips, etc.

FIG. 28 shows the heat-exchanging module 147 of the instrument intowhich the reaction vessel 40 is inserted for thermal processing andoptical detection of target analyte(s) in the reaction mixture. Themodule 147 preferably includes a housing 208 for holding the variouscomponents of the module. The module 147 also includes the thermalplates 190 described above. The housing 208 includes a slot (not shownin FIG. 28) above the plates 190 so that the reaction chamber of thevessel 40 may be inserted through the slot and between the plates. Theheat-exchanging module 147 also preferably includes a cooling system,such as a fan 212. The fan 212 is positioned to blow cooling air pastthe surfaces of the plates 190 to cool the plates and hence cool thereaction mixture in the vessel 40. The housing 208 preferably defineschannels for directing the cooling air past the plates 190 and out ofthe module 147.

The heat-exchanging module 147 further includes an optical excitationassembly 216 and an optical detection assembly 218 for opticallyinterrogating the reaction mixture contained in the vessel 40. Theexcitation assembly 216 includes a first circuit board 220 for holdingits electronic components, and the detection assembly 216 includes asecond circuit board 222 for holding its electronic components. Theexcitation assembly 216 includes one or more light sources (e.g., anLED. laser, or light bulb) for exciting fluorescently-labeled analytesin the vessel 40. The excitation assembly 216 also includes one or morelenses for collimating the light from the light sources, as well asfilters for selecting the excitation wavelength ranges of interest. Thedetection assembly 218 includes one or more detectors (e.g., aphotodiode, photomultiplier tube, or CCD) for detecting the lightemitted from the vessel 40. The detection assembly 218 also includes oneor more lenses for focusing and collimating the emitted light, as wellas filters for selecting the emission wavelength ranges of interest.Suitable optical excitation and detection assemblies for use in theheat-exchanging module 147 are described in International PublicationNumber WO 99/60380 (International Application Number PCT/US99/11182)published Nov. 25, 1999, the disclosure of which is incorporated byreference herein.

The optics assemblies 216, 218 are positioned in the housing 208 suchthat when the chamber of the vessel 40 is inserted between the plates190, the excitation assembly 216 is in optical communication with thechamber 42 through the optically transmissive side wall 57A (see FIG.22) and the detection assembly 218 is in optical communication with thechamber through the optically transmissive side wall 57B (FIG. 22). Inthe preferred embodiment, the optics assemblies 216, 218 are placed intooptical communication with the optically transmissive side walls bysimply locating the optics assemblies 216, 218 next to the bottom edgesof the plates 190 so that when the chamber of the vessel is placedbetween the plates, the optics assemblies 216, 218 directly contact, orare in close proximity to, the side walls.

FIG. 34 shows a partially cut-away, isometric view of the chamber of thevessel inserted between the plates 190A, 190B (the top portion of thevessel is cut away). The vessel preferably has an angled bottom portion(e.g., triangular) formed by the optically transmissive side walls 57A,57B. Each of the plates 190A, 190B has a correspondingly shaped bottomportion. The bottom portion of the first plate 190A has a first bottomedge 250A and a second bottom edge 2190B. Similarly, the bottom portionof the second plate 190B has a first bottom edge 252A and a secondbottom edge 252B. The first and second bottom edges of each plate arepreferably angularly offset from each other by the same angle that theside walls 57A, 57B are offset from each other (e.g., 90°).Additionally, the plates 190A, 190B are preferably positioned to receivethe chamber of the vessel between them such that the first side wall 57Ais positioned substantially adjacent and parallel to each of the firstbottom edges 250A, 252A and such that the second side wall 57B ispositioned substantially adjacent and parallel to each of the secondbottom edges 2190B, 252B. This arrangement provides for easy opticalaccess to the optically transmissive side walls 57A, 57B and hence tothe chamber of the vessel. A gel or fluid may optionally be used toestablish or improve optical communication between each optics assemblyand the side walls 57A, 57B. The gel or fluid should have a refractiveindex close to the refractive indexes of the elements that it iscoupling.

Referring again to FIG. 28, the optics assemblies 216, 218 arepreferably arranged to provide a 90° angle between excitation anddetection paths. The 90° angle between excitation and detection pathsassures that a minimum amount of excitation radiation entering throughthe first side wall of the chamber exits through the second side wall.Also, the 90° angle permits a maximum amount of emitted radiation to becollected through the second side wall. In the preferred embodiment, thevessel 40 includes a locating tab 58 (see FIG. 22) that fits into a slotformed between the optics assemblies 216, 218 to ensure properpositioning of the vessel 40 for optical detection. For improveddetection, the module 147 also preferably includes a light-tight lid(not shown) that is placed over the top of the vessel 40 and madelight-tight to the housing 208 after the vessel is inserted between theplates 190.

Although it is presently preferred to locate the optics assemblies 216,218 next to the bottom edges of the plates 190, many other arrangementsare possible. For example, optical communication may be establishedbetween the optics assemblies 216, 218 and the walls of the vessel 40via optical fibers, light pipes, wave guides, or similar devices. Oneadvantage of these devices is that they eliminate the need to locate theoptics assemblies 216, 218 physically adjacent to the plates 190. Thisleaves more room around the plates in which to circulate cooling air orrefrigerant, so that cooling may be improved.

The heat-exchanging module 147 also includes a PC board 226 for holdingthe electronic components of the module and an edge connector 224 forconnecting the module 147 to the instrument 140 (FIG. 10). The heatingelements and temperature sensors on the plates 190, as well as theoptical boards 220, 222, are connected to the PC board 226 by flexcables (not shown in FIG. 28 for clarity of illustration). The module147 may also include a grounding trace 228 for shielding the opticaldetection circuit. The module 147 may optionally include an indicator,such as an LED 214, for indicating to a user the current status of themodule such as “heating,” “cooling,” “finished,” or “fault”.

The housing 208 may be molded from a rigid, high-performance plastic, orother conventional material. The primary functions of the housing 208are to provide a frame for holding the plates 190, optics assemblies216, 218, fan 212, and PC board 226. The housing 208 also preferablyprovides flow channels and ports for directing cooling air from the fan212 across the surfaces of the plates 190 and out of the housing. In thepreferred embodiment, the housing 208 comprises complementary pieces(only one piece shown in the schematic side view of FIG. 28) that fittogether to enclose the components of the module 147 between them.

Referring again to FIG. 23, the plates 190A, 190B may be made of variousthermally conductive materials including ceramics or metals. Suitableceramic materials include aluminum nitride, aluminum oxide, berylliumoxide, and silicon nitride. Other materials from which the plates may bemade include, e.g., gallium arsenide, silicon, silicon nitride, silicondioxide, quartz, glass, diamond, polyacrylics, polyamides,polycarbonates, polyesters, polyimides, vinyl polymers, and halogenatedvinyl polymers, such as polytetrafluoroethylenes. Other possible platematerials include chrome/aluminum, superalloys, zircaloy, aluminum,steel, gold, silver, copper, tungsten, molybdenum, tantalum, brass,sapphire, or any of the other numerous ceramic, metal, or polymericmaterials available in the art.

Ceramic plates are presently preferred because their inside surfaces maybe conveniently machined to very high smoothness for high wearresistance, high chemical resistance, and good thermal contact to theflexible walls of the reaction vessel. Ceramic plates can also be madevery thin, preferably between about 0.6 and 1.3 mm, for low thermal massto provide for extremely rapid temperature changes. A plate made fromceramic is also both a good thermal conductor and an electricalinsulator, so that the temperature of the plate may be well controlledusing a resistive heating element coupled to the plate.

Various thermal elements may be employed to heat and/or cool the plates190A, 190B and thus control the temperature of the reaction mixture inthe chamber 42. In general, suitable heating elements for heating theplate include conductive heaters, convection heaters, or radiationheaters. Examples of conductive heaters include resistive or inductiveheating elements coupled to the plates, e.g., resistors orthermoelectric devices. Suitable convection heaters include forced airheaters or fluid heat-exchangers for flowing fluids past the plates.Suitable radiation heaters include infrared or microwave heaters.Similarly, various cooling elements may be used to cool the plates. Forexample, various convection cooling elements may be employed such as afan, peltier device, refrigeration device, or jet nozzle for flowingcooling fluids past the surfaces of the plates. Alternatively, variousconductive cooling elements may be used, such as a heat sink, e.g. acooled metal block, in direct contact with the plates.

Referring to FIG. 24, each plate 190 preferably has a resistive heatingelement 206 disposed on its outer surface. The resistive heating element206 is preferably a thick or thin film and may be directly screenprinted onto each plate 190, particularly plates comprising a ceramicmaterial, such as aluminum nitride or aluminum oxide. Screen-printingprovides high reliability and low cross-section for efficient transferof heat into the reaction chamber. Thick or thin film resistors ofvarying geometric patterns may be deposited on the outer surfaces of theplates to provide more uniform heating, for example by having denserresistors at the extremities and thinner resistors in the middle.Although it is presently preferred to deposit a heating element on theouter surface of each plate, a heating element may alternatively bebaked inside of each plate, particularly if the plates are ceramic. Theheating element 206 may comprise metals, tungsten, polysilicon, or othermaterials that heat when a voltage difference is applied across thematerial. The heating element 206 has two ends which are connected torespective contacts 204 which are in turn connected to a voltage source(not shown in FIG. 24) to cause a current to flow through the heatingelement. Each plate 190 also preferably includes a temperature sensor192, such as a thermocouple, thermistor, or RTD, which is connected bytwo traces 202 to respective ones of the contacts 204. The temperaturesensor 192 is be used to monitor the temperature of the plate 190 in acontrolled feedback loop.

The plates have a low thermal mass to enable rapid heating and coolingof the plates. In particular, it is presently preferred that each of theplates has a thermal mass less than about 5 J/° C., more preferably lessthan 3 J/° C., and most preferably less than 1 J/° C. As used herein,the term thermal mass of a plate is defined as the specific heat of theplate multiplied by the mass of the plate. In addition, each plateshould be large enough to cover a respective major wall of the reactionchamber. In the presently preferred embodiment, for example, each of theplates has a width X in the range of 2 to 22 mm, a length Y in the rangeof 2 to 22 mm, and a thickness in the range of 0.5 to 5 mm. The width Xand length Y of each plate is selected to be slightly larger than thewidth and length of the reaction chamber. Moreover, each platepreferably has an angled bottom portion matching the geometry of thebottom portion of the reaction chamber, as previously described withreference to FIG. 34. Also in the preferred embodiment, each of theplates is made of aluminum nitride having a specific heat of about 0.75J/g° C. The mass of each plate is preferably in the range of 0.005 to5.0 g so that each plate has a thermal mass in the range of 0.00375 to3.75 J/° C.

The opposing plates 190 are positioned to receive the chamber of thevessel 40 between them such that the flexible major walls of the chambercontact and conform to the inner surfaces of the plates. It is presentlypreferred that the plates 190 be held in an opposing relationship toeach other using, e.g., brackets, supports, or retainers. Alternatively,the plates 190 may be spring-biased towards each other as described inInternational Publication Number WO 98/38487, the disclosure of which isincorporated by reference herein. In another embodiment of theinvention, one of the plates is held in a fixed position, and the secondplate is spring-biased towards the first plate. If one or more springsare used to bias the plates towards each other, the springs should besufficiently stiff to ensure that the plates are pressed against theflexible walls of the vessel with sufficient force to cause the walls toconform to the inner surfaces of the plates.

FIGS. 29-30 illustrate a preferred support structure 209 for holding theplates 190A, 190B in an opposing relationship to each other. FIG. 29shows an exploded view of the structure, and FIG. 30 shows an assembledview of the structure. For clarity of illustration, the supportstructure 209 and plates 190A, 190B are shown upside down relative totheir normal orientation in the heat-exchanging module of FIG. 28.Referring to FIG. 29, the support structure 209 includes a mountingplate 210 having the slot 148 formed therein. The slot 148 issufficiently large to enable the chamber of the vessel to be insertedthrough it. Spacing posts 230A, 230B extend from the mounting plate 210on opposite sides of the slot 148. Spacing post 230A has indentations232 formed on opposite sides thereof (only one side visible in theisometric view of FIG. 29), and spacing post 230B has indentations 234formed on opposite sides thereof (only one side visible in the isometricview of FIG. 29). The indentations 232, 234 in the spacing posts are forreceiving the edges of the plates 190A, 190B. To assemble the structure,the plates 190A, 190B are placed against opposite sides of the spacingposts 230A, 230B such that the edges of the plates are positioned in theindentations 232, 234. The edges of the plates are then held in theindentations using a suitable retention means. In the preferredembodiment, the plates are retained by retention clips 236A, 236B.Alternatively, the plates 190A, 190B may be retained by adhesive bonds,screws, bolts, clamps, or any other suitable means.

The mounting plate 210 and spacing posts 230A, 2301B are preferablyintegrally formed as a single molded piece of plastic. The plasticshould be a high temperature plastic, such as polyetherimide, which willnot deform of melt when the plates 190A, 190B are heated. The retentionclips 230A, 230B are preferably stainless steel. The mounting plate 210may optionally include indentations 240A, 240B for receiving flex cables238A, 238B, respectively, that connect the heating elements andtemperature sensors disposed on the plates 190A, 190B to the PC board226 of the heat-exchanging module 147 (FIG. 28). The portion of the flexcables 238A adjacent the plate 190A is held in the indentation 240A by apiece of tape 242A, and the portion of the flex cables 238B adjacent theplate 190B is held in the indentation 240B by a piece of tape 242B.

FIG. 31 is an isometric view of the assembled support structure 209. Themounting plate 210 preferably includes tabs 246 extending from oppositesides thereof for securing the structure 209 to the housing of theheat-exchanging module. Referring again to FIG. 28, the housing 208preferably includes slots for receiving the tabs to hold the mountingplate 210 securely in place. Alternatively, the mounting plate 210 maybe attached to the housing 208 using, e.g., adhesive bonding, screws,bolts, clamps, or any other conventional means of attachment.

Referring again to FIG. 29, the support structure 209 preferably holdsthe plates 190A, 190B so that their inner surfaces are angled veryslightly towards each other. In the preferred embodiment, each of thespacing posts 230A, 230B has a wall 244 that is slightly tapered so thatwhen the plates 190A, 190B are pressed against opposite sides of thewall, the inner surfaces of the plates are angled slightly towards eachother. As best shown in FIG. 23, the inner surfaces of the plates 190A,190B angle towards each other to form a slightly V-shaped slot intowhich the chamber 42 is inserted. The amount by which the inner surfacesare angled towards each other is very slight, preferably about 1° fromparallel. The surfaces are angled towards each other so that, prior tothe insertion of the chamber 42 between the plates 190A, 190B, thebottoms of the plates are slightly closer to each other than the tops.This slight angling of the inner surfaces enables the chamber 42 of thevessel to be inserted between the plates and withdrawn from the platesmore easily. Alternatively, the inner surfaces of the plates 190A, 190Bcould be held parallel to each other, but insertion and removal of thevessel 40 would be more difficult.

In addition, the inner surfaces of the plates 190A, 190B are preferablyspaced from each other a distance equal to the thickness of the frame46. In embodiments in which the inner surfaces are angled towards eachother, the centers of the inner surfaces are preferably spaced adistance equal to the thickness of the frame 46 and the bottoms of theplates are initially spaced a distance that is slightly less than thethickness of the frame 46. When the chamber 42 is inserted between theplates 190A, 190B, the rigid frame 46 forces the bottom portions of theplates apart so that the chamber 42 is firmly sandwiched between theplates. The distance that the plates 190A, 190B are wedged apart by theframe 46 is usually very small, e.g., about 0.035 mm if the thickness ofthe frame is 1 mm and the inner surfaces are angled towards each otherby 1°.

Referring again to FIG. 30, the retention clips 236A, 236B should besufficiently flexible to accommodate this slight outward movement of theplates 190A, 190B, yet sufficiently stiff to hold the plates within therecesses in the spacing posts 230A, 230B during insertion and removal ofthe vessel. The wedging of the vessel between the plates 190A, 190Bprovides an initial preload against the chamber and ensures that theflexible major walls of the chamber, when pressurized, establish goodthermal contact with the inner surfaces of the plates.

Referring again to FIG. 28, to limit the amount that the plates 190 canspread apart due to the pressurization of the vessel 40, stops may bemolded into the housings of optics assemblies 216, 218. As shown in FIG.32, the housing 249 of the optics assembly 218 includes claw-like stops247A, 247B that extend outwardly from the housing. As shown in FIG. 33,the housing 249 is positioned such that the bottom edges of the plates190A, 190B are inserted between the stops 247A, 247B. The stops 247A,247B thus prevent the plates 190A, 190B from spreading farther than apredetermined maximum distance from each other. Although not shown inFIG. 33 for illustrative clarity, the optics assembly 216 (see FIG. 28)has a housing with corresponding stops for preventing the other halvesof the plates from spreading farther than the predetermined maximumdistance from each other. Referring again to FIG. 23, the maximumdistance that stops permit the inner surfaces of the plates 190A, 190Bto be spaced from each other should closely match the thickness of theframe 46. Preferably, the maximum spacing of the inner surfaces of theplates 190A, 190B is slightly larger than the thickness of the frame 46to accommodate tolerance variations in the vessel 40 and plates 190A,190B. For example, the maximum spacing is preferably about 0.1 to 0.3 mmgreater than the thickness of the frame 46.

FIG. 35 is a schematic, block diagram of the electronic components ofthe heat-exchanging module 147. The module includes a connector 224 orflex cable for connection to the main logic board of the instrument. Themodule also includes heater plates 190A, 190B each having a resistiveheating element as described above. The plates 190A, 190B are wired inparallel to receive power input 253 from the instrument. The plates190A, 190B also include temperature sensors 192A, 192B that outputanalog temperature signals to an analog-to-digital converter 264. Theconverter 264 converts the analog signals to digital signals and routesthem to the microcontroller in the instrument through the connector 224.

The heat-exchanging module also includes a cooling system, such as a fan212, for cooling the plates 190A, 190B and the reaction mixturecontained in the vessel inserted between the plates. The fan 212 isactivated by switching a power switch 272, which is in turn controlledby a control logic block 270 that receives control signals from themicrocontroller. The module further includes four light sources, such asLEDs 200, for excitation of labeled analytes in the reaction mixture andfour detectors 198, preferably photodiodes, for detecting fluorescentemissions from the reaction mixture. The module also includes anadjustable current source 255 for supplying a variable amount of current(e.g., in the range of 0 to 30 mA) to each LED to vary the brightness ofthe LED. A digital-to-analog converter 260 is connected between theadjustable current source 255 and the microcontroller to permit themicrocontroller to adjust the current source digitally.

The adjustable current source 255 is preferably used to ensure that eachLED has about the same brightness when activated. Due to manufacturingvariances, many LEDs have different brightnesses when provided with thesame amount of current. Therefore, it is presently preferred to test thebrightness of each LED during manufacture of the heat-exchanging moduleand to store calibration data in a memory 268 of the module. Thecalibration data indicates the correct amount of current to provide toeach LED. The microcontroller reads the calibration data from the memory268 and controls the current source 255 accordingly.

The module additionally includes a signal conditioning/gainselect/offset adjust block 262 comprised of amplifiers, switches,electronic filters, and a digital-to-analog converter. The block 262adjusts the signals from the detectors 198 to increase gain, offset, andreduce noise. The microcontroller controls block 262 through a digitaloutput register 266. The output register 266 receives data from themicrocontroller and outputs control voltages to the block 262. The block262 outputs the adjusted detector signals to the microcontroller throughthe analog-to-digital converter 264 and the connector 224. The modulealso includes the memory 268, preferably a serial EEPROM, for storingdata specific to the module, such as calibration data for the LEDs 200,thermal plates 190A, 190B, and temperature sensors 192A, 192B.

The operation of the cartridge and instrument will now be described. Asshown in FIG. 3, a fluid sample to be analyzed is added to the samplechamber 65 through the sample port 64 and the cap 30 screwed into theport 64 to seal the port shut. Referring to FIG. 10, the cartridge 20 isthen placed into the cartridge nest 141 of the instrument 140 forprocessing. All valves in the cartridge 20 are initially closed when thecartridge is placed into the instrument 140. When the cartridge isplaced in the instrument, the transducer 92 contacts an external surfaceof the flexible gasket 63 forming the bottom wall of the lysing chamber86, as shown in FIG. 5.

Referring again to FIG. 10, the instrument 140 is preferablycomputer-controlled to perform the functions described in the followingsection, e.g., opening and closing valves in the cartridge using valveactuators 142, providing pressure to the cartridge through nozzles 145,activating the transducer 92, sensing liquid presence or liquid levelsusing optical sensors 143 and 144, and controlling the heat-exchangingand optical detection module 147. A programmer having ordinary skill inthe art will be able to program a microcontroller and/or computer toperform these functions based upon the following description.

Referring to FIG. 9, liquids are preferably forced to flow through thecartridge using differential pressure. Although positive pressure isdescribed herein, negative pressure (vacuum) may also be used to controlfluid flow in the cartridge. The maximum amount of positive pressurethat can be applied is usually limited by the hydrophobic membraneswhich may reach liquid break-through pressure above 30 pounds per squareinch (psi). The lower limit of pressure is limited by the need to movesample and other fluids through the cartridge sufficiently quickly tomeet assay goals. Below 1 psi, for example, sample may not flowefficiently through the filter stack 87. Pressure in the range of 6 to20 psi is generally adequate. The sample flow rate through the cartridgeis preferably in the range of 10 to 30 ml/minute. The wash flow rate maybe slower, e.g. 6 to 18 ml/minute so that the wash effectively washesthe lysing chamber 86.

A specific protocol will now be described with reference to FIG. 9 toillustrate the operation of the cartridge. It is to be understood thatthis is merely an example of one possible protocol and is not intendedto limit the scope of the invention. To begin, the cartridge ispreferably primed with wash solution from the wash chamber 66 before thefluid sample is forced to flow from the sample chamber 65. To prime thecartridge, valves 111 and 115 are opened and a pressure of 10 psi isapplied to the chamber 66 through the pressure port 116 for about twoseconds. A small portion of the wash solution flows through the channels117 and 106, through the lysing chamber 86, through the channels 109 and110, into the U-shaped channel 122, and all the way to the hydrophobicmembrane below the pressure port 128.

Following priming, valve 115 and pressure port 116 are closed and valves107 and 114 are opened. At the same time, a pressure of 20 psi isapplied to the sample chamber 65 through the pressure port 105 for about15 seconds to force the sample to flow through the channel 106, throughthe filter stack 87 in the chamber 87, through the channels 110, 111,112 and into the vented waste chamber 68. As the sample passes thedetection region 136 in the channel 106, the reflective optical sensor144 (FIG. 13) may be used to determine when the sample chamber 65 hasbeen emptied. As the sample liquid flows through the filter stack 87,target cells or viruses in the sample are captured. When a predeterminedvolume of sample reaches the waste chamber 68, some of the liquid spillsover into the sensor chamber 120, triggering the next step in theprotocol. Alternatively, instead of using feedback from optical sensorsto trigger events, the steps in a predetermined protocol may simply betimed, e.g., applying predetermined pressures for predetermineddurations of time to move known volumes of fluid at known flow rates.

The flow-through design of the lysing chamber 86 permits target cells orviruses from a relatively large sample volume to be concentrated into amuch smaller volume for amplification and detection. This is importantfor the detection of low concentration analyte in the sample, such asnucleic acid. In particular, the ratio of the volume of the sampleforced to flow through the lysing chamber 86 to the volume capacity ofthe chamber 86 is preferably at least 2:1, and more preferably at least5:1. The volume of sample forced to flow through the chamber 86 ispreferably at least 100 □l, and more preferably at least 1 ml. In thepresently preferred embodiment, a sample volume of 5 ml is forced toflow through the lysing chamber 86, and the chamber 86 has a volumecapacity of about 0.5 ml, so that the ratio is 10:1. In addition, thelysing chamber 86 may be sonicated (e.g., using an ultrasonic horncoupled to a wall of the chamber) as the sample is forced to flowthrough the chamber. Sonicating the chamber 86 helps to prevent cloggingof the filter stack 87, providing for more uniform flow through thechamber 86. In particular, the sound waves help keep particulate matteror the beads in the filter stack from clogging one or more filters.

In the next step, valves 111, 114, 115 are opened and a pressure of 20psi is applied to the wash chamber 66 for about seven seconds to forcethe wash solution to flow through the channels 117 and 106 into thelysing chamber 86. The washing solution washes away PCR inhibitors andcontaminants from the lysing chamber 86 and carries then through thechannels 109, 110, and 112 into the waste chamber 68. A variety ofsuitable wash solutions of varying pH, solvent composition, and ionicstrength may be used for this purpose and are well known in the art. Forexample, a suitable washing reagent is a solution of 80 mM potassiumacetate, 8.3 mM Tris-HCl, pH 7.5, 40 uM EDTA, and 55% ethanol. Thelysing chamber 86 may be sonicated (e.g., using an ultrasonic horncoupled to a wall of the chamber) while the wash solution is forced toflow through the chamber. Sonicating the chamber 86 helps to preventclogging of the filter stack 87, providing for more uniform flow throughthe chamber 86 as previously described. In addition, the sound waves mayhelp loosen the material to be washed away. When the incremental volumeof wash solution reaches the waste chamber 68, some of the liquid spillsover into the sensor chamber 121, triggering the next step in theprotocol.

In the next step, valve 115 is closed and valve 119 is opened while apressure of 15 psi is applied to the reagent chamber 67 through thepressure port 118 for about three seconds. The pressure forces lysingreagent to flow from the chamber 67 through the channels 117, 106 intothe lysing chamber 86, and into the channel 110. The chamber 86 is thusfilled with liquid. Suitable lysing reagents include, e.g., solutionscontaining a chaotropic salt, such as guanidine HCl, guanidinethiocyanate, guanidine isothiocyanate, sodium iodide, urea, sodiumperchlorate, and potassium bromide. In the presently preferredembodiment, a lysing reagent that is not inhibitory to PCR is used. Thelysing reagent comprises 10 mM tris, 5% tween-20, 1 mM tris(2-carboxyethyl phosphine hydrochloride), 0.1 mM Ethylene Glycol-bis(b-amino-ethyl ether)-N,N,N′,N′-tetracetic acid. After the lysingchamber 86 is filled with lysing reagent, the valves 111, 114 areclosed. Valve 119 remains open and a pressure of 20 psi is applied topressure port 118. The static pressure in the lysis chamber 86 istherefore increased to 20 psi in preparation for the lysis of the cellsor viruses trapped in the filter stack 87.

Referring again to FIG. 5, the pressurization of the lysing chamber 86is important because it ensures effective coupling between thetransducer 92 and the flexible wall 63 of the lysing chamber 86. Todisrupt the cells or viruses in the chamber 86, the transducer 92 isactivated (i.e., set into vibratory motion). The flexible wall 63 of thelysing chamber 86 transfers the vibratory motion of the transducer 92 tothe liquid in the chamber 86 by allowing slight deflections withoutcreating high stresses in the wall. The wall 63 may be formed by theelastomeric membrane as previously described. Alternatively, the wallmay be a film or sheet of polymeric material (e.g., a polypropylenefilm) preferably having a thickness in the range of 0.025 to 0.1 mm. Thetransducer 92 is preferably an ultrasonic horn for sonicating thechamber 86. The chamber 86 is preferably sonicated for 10 to 40 secondsat a frequency in the range of 20 to 60 kHz. In the exemplary protocol,the chamber is sonicated for 15 seconds at a frequency of 47 kHz. Theamplitude of the horn tip is preferably in the range of 20 to 25 □m(measured peak to peak).

As the tip of the transducer 92 vibrates, it repeatedly impacts theflexible wall 63. On its forward stroke (in the upward direction in FIG.6), the tip of the transducer 92 pushes the wall 63 and creates apressure pulse or pressure wave in the chamber 86. On its retreatingstroke (downward in FIG. 5), the tip of the transducer 92 usuallyseparates from the flexible wall 63 because the flexible wall 63 cannotmove at the same frequency as the transducer. On its next forwardstroke, the tip of the transducer 92 once again impacts the wall 63 in ahead-on collision as the tip and wall speed towards each other. Becausethe transducer 92 and the wall 63 separate as the transducer 92vibrates, the effective forward stroke of the transducer is less thanits peak-to-peak amplitude. The effective forward stroke determines thelevel of sonication in the chamber 86. It is therefore important toincrease the static pressure in the lysing chamber 86 so that when thetip of the transducer 92 retreats, the flexible wall 63 is forcedoutwardly to meet the tip on its return stroke. The static pressure inthe chamber 86 should be sufficient to ensure that the effective forwardstroke of the transducer 92 generates pressure pulses or pressure wavesin the chamber 86. It is presently preferred to increase the staticpressure in the chamber 86 to at least 5 psi above the ambient pressureexternal to the cartridge, and more preferably to a pressure in therange of 15 to 25 psi above the ambient pressure.

On each forward stroke, the transducer 92 imparts a velocity to theliquid in the chamber 86, thus creating a pressure pulse that quicklysweeps across the chamber 86. The beads in the filter stack 87 (FIG. 6)are agitated by the pressure pulses in the chamber 86. The pressurepulses propel the beads into violent motion in the chamber 86, and thebeads mechanically rupture the cells or viruses to release the material(e.g., nucleic acid) therefrom. It should be noted that some types ofcells, such as blood cells, are relatively weak and may be disruptedusing only pressure pulses without the use of beads. Other types ofcells (particularly spores) have highly resistant cell walls and beadsare generally required for effective lysis.

Referring again to FIG. 9, following disruption of the cells or viruses,valves 111, 124 are opened and a pressure of 12 psi is delivered forabout 4 seconds to the reagent chamber 67 through the pressure port 118.The pressure forces the lysis reagent to elute the nucleic acid from thefilter stack 87 and to flow with the nucleic acid into theneutralization chamber 70. The lysing chamber 86 may be sonicated (e.g.,using an ultrasonic horn coupled to a wall of the chamber) while theeluting the nucleic acid. Sonicating the chamber 86 may help preventclogging of the filter stack 87, as previously described. The chamber420 is partially filled (e.g., half-filled) with neutralizer, such asdetergent, for neutralizing the lysing reagent. If a lysing reagentnon-inhibitory to PCR is used, the neutralizer is optional.

In the next step, the valve 124 is closed to hold the lysing reagent,analyte, and neutralizer in the chamber 70. The valve 114 is opened anda pressure of 15 psi is applied for about three seconds through thepressure port 128 to force any liquid in the U-shaped channel 122 toflow into the waste chamber 68. Next, valves 124 and 126 are opened anda pressure of 15 psi is applied for about five seconds through thepressure port 123 on top of the neutralizer chamber 70. The pressureforces the neutralized lysing reagent and nucleic acid in the chamber 70to flow into the channel 122 and into the master mix chamber 71. Thevalve 126 to the master mix chamber 71 is then closed. The master mixchamber contains PCR reagents and fluorescent probes that mix with theneutralized lysing reagent and nucleic acid to form a reaction mixture.

In the next step, the channel 122 is cleared by opening valve 114 towaste chamber 68 and applying a pressure of 15 psi for about one secondto pressure port 128. In the next step, the reaction mixture formed inthe master mix chamber 71 is moved into the reaction vessel 40 asfollows. Valves 126, 127, and 133 are opened and a pressure of 15 psi isapplied for about six seconds to the pressure port 125 on top of themaster mix chamber 71 to force the reaction mixture to flow through thechannel 122, valve 127, and channel 80 into the reaction vessel 40through the port 41. The reaction mixture fills the chamber 42 of thevessel, displacing air in the chamber which exits through the outletchannel 52. The air escaping through the outlet channel 52 travels inchannel 81 past sensor region 130 and into channel 131. From channel131, the air flows into channel 132, through valve 133, channel 134, andexits the cartridge through the vent 36. When a volume of reactionmixture sufficient to fill the chamber 42 has flowed into the vessel,excess reaction mixture exits the vessel through the outlet channel 52.The excess reaction mixture flows into channel 81 and is opticallydetected in the sensor region 130. When the reaction mixture isdetected, valve 133 is closed while pressure from the pressure port 125is applied to pressurize the reaction chamber 42.

Referring again to FIG. 23, the pressurization of the chamber 42 expandsthe flexible major walls 48 of the vessel. In particular the pressureforces the major walls 48 to contact and conform to the inner surfacesof the plates 190A, 190B. This ensures optimal thermal conductancebetween the plates 190A, 190B and the reaction mixture in the chamber42. It is presently preferred to pressurize the chamber 42 to a pressurein the range of 2 to 30 psi above ambient pressure. This range ispresently preferred because 2 psi is generally enough pressure to ensureconformity between the walls 48 and the surfaces of the plates 190A,190B, while pressures above 30 psi may cause bursting of the walls 48,deformation of the frame 46 or plates 190A, 190B, or bursting of thehydrophobic membranes in the cartridge. More preferably, the chamber 42is pressurized to a pressure in the range of 8 to 15 psi above ambientpressure. This range is more preferred because it is safely within thepractical limits described above. When the chamber 42 is pressurized,the reaction mixture in the vessel 40 is thermally processed andoptically interrogated to determine the presence or absence of a targetanalyte in the mixture.

Referring again to FIG. 35, the reaction mixture is thermally processedbetween the plates 190A, 190B using standardproportional-integral-derivative (PID) control using target temperaturesand feedback signals from the temperature sensors 192A, 192B.Proportioning may be accomplished either by varying the ratio of “on”time to “off” time, or, preferably with proportional analog outputswhich decrease the average power being supplied either to the heatingelements on the plates 190A, 190B or to the fan 212 as the actualtemperature of the plates 190A, 190B approaches the desired set pointtemperature. PID control combines the proportional mode with anautomatic reset function (integrating the deviation signal with respectto time) and rate action (summing the integral and deviation signal toshift the proportional band). Standard PID control is well known in theart and need not be described further herein. Alternatively, thereaction mixture may be thermally processed using a modified version ofPID control described in International Publication Number WO 99/48608(Application Number PCT/US99/06628) the disclosure of which isincorporated by reference herein.

As the reaction mixture is thermally cycled between the heater plates190A, 190B to amplify one or more target nucleic acid sequences in themixture, the mixture is optically interrogated, preferably at the lowesttemperature point in each cycle. Optical interrogation is accomplishedby sequentially activating each of the LEDs 200 to excite differentfluorescently-labeled analytes in the mixture and by detecting lightemitted (fluorescent output) from the chamber 42 using detectors the198. Referring again to FIG. 22, excitation beams are preferablytransmitted to the chamber 42 through the optically transmissive sidewall 57A, while fluorescent emission is detected through the side wall57B.

One advantage of the cartridge of the present invention is that itallows the intracellular material from a relatively large volume offluid sample, e.g. several milliliters or more, to be separated from thesample and concentrated into a much smaller volume of reaction fluid,e.g., 100 μl or less. The cartridge permits extraordinary concentrationfactors by efficiently extracting material from milliliter quantities offluid sample. In particular, the sample chamber 65 preferably has avolume capacity in the range of 100 μl to 12 ml. More preferably, thesample chamber 65 has a volume capacity of at least 1 ml. The lowerlimit of 1 ml is preferred because at least 1 ml of sample should beanalyzed to detect low concentration analytes such as nucleic acid. Theupper limit of 12 ml is preferred because a sample volume greater than12 ml would require a much larger cartridge and likely clog the filterstack. In the presently preferred embodiment, the sample chamber has avolume capacity of 5.5 ml for holding 5 ml of sample.

The wash chamber 66 has a volume capacity proportional to the volume ofthe lysing chamber 86. In particular, the wash chamber 66 preferablyholds a volume of wash that is at least one to two times the volume ofthe lysing chamber 86 to ensure that there is enough wash solution towash out PCR inhibitors and debris from the chamber 86. In the presentlypreferred embodiment, the volume of the lysing chamber 86 is about 0.5ml and the volume of the wash chamber 66 is 2.5 ml for holding 2 ml ofwash solution. The lysing chamber volume of 0.5 ml is a compromisebetween a size large enough to avoid clogging of the filter stack 87 anda size small enough to concentrate analyte into a small volume forimproved amplification and detection.

The reagent chamber 67 preferably holds a volume of lysing reagent thatis at least one to two times the volume of the lysing chamber 86 so thatthere is sufficient lysing reagent to pressurize the chamber and toelute nucleic acid from the chamber. In the presently preferredembodiment, the chamber 67 has a volume capacity of 1.5 ml for holdingabout 1 to 1.5 ml of lysing reagent. The waste chamber 68 has a volumecapacity sufficient to hold the sample, wash solution, and unused lysingreagent. The waste chamber 68 is sized at 9.5 ml volume capacity in thepreferred embodiment.

The size of the neutralization chamber 70 is dependent upon the volumeof the lysing chamber 86 since the neutralizer in the chamber 70neutralizes the volume of lysing reagent that fills the lysing chamber86. It is currently preferred that the lysing chamber have a volume if0.5 ml, so the chamber 70 has a volume capacity of 1.0 ml for holdingabout 0.5 ml of neutralizer that is mixed with 0.5 ml of the lysingreagent and eluted analyte. The volume capacity of the master mixchamber 71 should be sufficient to produce a reaction mixture to fillthe vessel 40 and the channels 122, 127 leading to the vessel. In thepresently preferred embodiment, the master mix chamber has a volumecapacity of 200 μl for holding an initial load of 100 μl of master mixto which is added 100 μl of neutralized lysing reagent and elutedanalyte to form the reaction mixture.

The flow channels in the cartridge are generally D-shaped in crosssection (with the gasket 63 forming the flat side of the channel) andpreferably have a width or diameter in the range of 1/64 to ⅛ of an inch(0.4 to 3.2 mm), and more preferably a width of 1/32 to 1/16 of an inch(0.8 to 1.6 mm). These ranges are presently preferred to avoid havingchannels to narrow (which creates flow restriction) and to avoid havingchannels too wide (which yields unused volumes of liquid sitting in theflow path).

Many modifications to the structure and operation of the cartridge andinstrument are possible in alternative embodiments. For example,although amplification by PCR is presently preferred, the cartridge andinstrument may be used to amplify nucleic acid sequences using anyamplification method, including both thermal cycling amplificationmethods and isothermal amplification methods. Suitable thermal cyclingmethods include, but are not limited to, the Polymerase Chain Reaction(PCR; U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,965,188); ReverseTranscriptase PCR (RT-PCR); DNA Ligase Chain Reaction (LCR;International Patent Application No. WO 89/09835); andtranscription-based amplification (D. Y. Kwoh et al. 1989, Proc. Natl.Acad. Sci. USA 86, 1173-1177). Suitable isothermal amplification methodsuseful in the practice of the present invention include, but are notlimited to, Rolling Circle Amplification; Strand DisplacementAmplification (SDA; Walker et al. 1992, Proc. Natl. Acad. Sci. USA 89,392-396); Q-.beta. replicase (Lizardi et al. 1988, Bio/Technology 6,1197-1202); Nucleic Acid-Based Sequence Amplification (NASBA; R.Sooknanan and L. Malek 1995, Bio/Technology 13, 563-65); andSelf-Sustained Sequence Replication (3 SR; Guatelli et al. 1990, Proc.Natl. Acad. Sci. USA 87, 1874-1878).

Moreover, the cartridge and instrument may be used to conduct chemicalreactions other than nucleic acid amplification. Further, althoughfluorescence excitation and emission detection is preferred, opticaldetection methods such as those used in direct absorption and/ortransmission with on-axis geometries may also be used to detect analytein the cartridge. Another possible detection method is time decayfluorescence. Additionally, the cartridge is not limited to detectionbased upon fluorescent labels. For example, detection may be based uponphosphorescent labels, chemiluminescent labels, orelectrochemiluminescent labels.

A fluid sample may be introduced into the cartridge by a variety ofmeans, manual or automated. For manual addition, a measured volume ofmaterial may be placed into a receiving area of the cartridge through aninput port and a cap is then placed over the port. Alternatively, agreater amount of sample material than required for the analysis can beadded to the cartridge and mechanisms within the cartridge can effectthe precise measuring and aliquoting of the sample needed for thespecified protocol. It may be desirable to place certain samples, suchas tissue biopsy material, soil, feces, exudates, and other complexmaterial into another device or accessory and then place the secondarydevice or accessory into the cartridge. For example, a piece of tissuemay be placed into the lumen of a secondary device that serves as thecap to the input port of the cartridge. When the cap is pressed into theport, the tissue is forced through a mesh that slices or otherwisedivides the tissue.

For automated sample introduction, additional design features of thecartridge are employed and, in many cases, impart specimen accessionfunctionality directly into the cartridge. With certain samples, such asthose presenting a risk of hazard to the operator or the environment,such as human retrovirus pathogens, the transfer of the sample to thecartridge may pose a risk. Thus, in one embodiment, a syringe may beintegrated into a device to provide a means for moving external fluidicsamples directly into the cartridge. Alternatively, a venous punctureneedle and an evacuated blood tube can be attached to the cartridgeforming an assembly that can be used to acquire a sample of blood. Aftercollection, the tube and needle are removed and discarded, and thecartridge is then placed in an instrument to effect processing. Theadvantage of such an approach is that the operator or the environment isnot exposed to pathogens.

The input port can be designed with a consideration of appropriate humanfactors as a function of the nature of the intended specimen. Forexample, respiratory specimens may be acquired from the lowerrespiratory tract as expectorants from coughing, or as swab or brushsamples from the back of the throat or the nares. In the former case,the input port can be designed to allow the patient to cough directlyinto the cartridge or to otherwise facilitate spitting of theexpectorated sample into the cartridge. For brush or swab specimens, thespecimen is placed into the input port where features of the port andclosure facilitate the breaking off and retaining of the end of the swabor brush in the cartridge receiving area.

In another embodiment, the cartridge includes input and output tubesthat may be positioned in a sample pool of very large volume, such as aflowing stream of water, so that the sample material flows through thecartridge. Alternatively, a hydrophilic wicking material can serve as aninteractive region so that the entire cartridge can be immersed directlyinto the specimen, and a sufficient amount of specimen is absorbed intothe wicking material. The cartridge is then removed, and can betransported to the laboratory or analyzed directly using a portableinstrument. In another embodiment, tubing can be utilized so that oneend of the tube is in direct communication with the cartridge to providea fluidic interface with at least one interactive region and the otherend is accessible to the external environment to serve as a receiver forsample. The tube can then be placed into a specimen and serve as asipper. The cartridge itself may also serve as the actual specimencollection device, thereby reducing handling and inconvenience. In thecase of specimens involved in legal disputes or criminal investigations,the direct accessing of the test material into the fluidic cartridge isadvantageous because the chain of custody is conveniently and reliablypreserved.

Referring again to FIG. 9, reagents may be exogenously introduced intothe cartridge before use, e.g., through sealable openings in the reagentchamber 67, neutralizer chamber 70, and master mix chamber 71.Alternatively, the reagents may be placed in the cartridge duringmanufacture, e.g., as aqueous solutions or dried reagents requiringreconstitution. The particular format is selected based on a variety ofparameters, including whether the interaction is solution-phase orsolid-phase, the inherent thermal stability of the reagent, speed ofreconstitution, and reaction kinetics. Reagents containing compoundsthat are thermally unstable when in solution can be stabilized by dryingusing techniques such as lyophilization. Additives, such as simplealcohol sugars, methylcelluloses, and bulking proteins may be added tothe reagent before drying to increase stability or reconstitutability.

Referring again to FIG. 21, the reaction vessel 40 does not require twoflexible sheets forming opposing major walls 48 of the reaction chamber42. For example, in one alternative embodiment, the vessel 40 has onlyone flexible sheet forming a major wall of the chamber. The rigid frame46 defines the other major wall of the chamber, as well as the sidewalls of the chamber. In this embodiment, the major wall formed by theframe 46 should have a minimum thickness of about 0.05 inches (1.25 mm)which is typically the practical minimum thickness for injectionmolding, while the flexible sheet may be as thin as 0.0005 inches(0.0125 mm). The advantage to this embodiment is that the manufacturingof the reaction vessel 40 is simplified, and hence less expensive, sinceonly one flexible sheet need be attached to the frame 46. Thedisadvantage is that the heating and cooling rates of the reactionmixture are likely to be slower since the major wall formed by the frame46 will probably not permit as high a rate of heat transfer as the thin,flexible sheet.

Referring to FIG. 28, the heat-exchanging module 147 only requires onethermal surface for contacting a flexible wall of the reaction vessel 40and one thermal element for heating and/or cooling the thermal surface.The advantage to using one thermal surface and one thermal element isthat the apparatus may be manufactured less expensively. Thedisadvantage is that the heating and cooling rates are likely to beabout twice as slow. Further, although it is presently preferred thatthe thermal surfaces be formed by the thermally conductive plates 190,each thermal surface may be provided by any rigid structure having acontact area for contacting a wall of the vessel 40. The thermal surfacepreferably comprises a material having a high thermal conductivity, suchas ceramic or metal. Moreover, the thermal surface may comprise thesurface of the thermal element itself. For example, the thermal surfacemay be the surface of a thermoelectric device that contacts the wall toheat and/or cool the chamber.

It is presently preferred to build the transducer into the instrument140. In another embodiment, however, the transducer may be built intothe cartridge. For example, a piezoelectric disk may be built into thecartridge for sonicating the lysing chamber. Alternatively, a speaker orelectromagnetic coil device may be built into the cartridge. In theseembodiments, the cartridge includes suitable electrical connectors forconnecting the transducer to a power supply. In embodiments in which thetransducer is built into the cartridge, the transducer should beprevented from contacting the fluid sample directly, e.g., thetransducer should be laminated or separated from the sample by a chamberwall. Further, lysis of the cells or viruses may be performed using aheater in place of or in combination with a transducer. The heater maybe a resistive heating element that is part of cartridge, or the heatercould be built into the instrument that receives the cartridge. In thisembodiment, the cells or viruses are disrupted by heating the lysischamber to a high temperature (e.g., 95° C.) to disrupt the cell walls.

FIGS. 36-46 show another apparatus 350 for disrupting cells or virusesaccording to the present invention. FIG. 36 shows an isometric view ofthe apparatus 350, and FIG. 37 shows a cross sectional view of theapparatus 350. As shown in FIGS. 36-37, the apparatus 350 includes acartridge or container 358 having a chamber 367 for holding the cells orviruses. The container includes a flexible wall 440 defining the chamber367. In this embodiment, the flexible wall 440 is the bottom wall of thechamber 367. The flexible wall 440 is preferably a sheet or film ofpolymeric material (e.g., a polypropylene film) and the wall 440preferably has a thickness in the range of 0.025 to 0.1 mm. Theapparatus 350 also includes a transducer 314, such as an ultrasonichorn, for contacting an external surface of the flexible wall 440 (i.e.,a surface of the wall 440 that is external to the chamber 367). Thetransducer 314 should be capable of vibratory motion sufficient tocreate pressure pulses in the chamber 367. Suitable transducers includeultrasonic, piezoelectric, magnetostrictive, or electrostatictransducers. The transducer may also be an electromagnetic device havinga wound coil, such as a voice coil motor or a solenoid device.

The apparatus 350 further includes a support structure 352 for holdingthe container 358 and the transducer 314 against each other such thatthe transducer 314 contacts the wall 440 of the chamber 367 and forapplying a substantially constant force to the container 358 or to thetransducer 314 to press together the transducer 314 and the wall 440 ofthe chamber. The support structure 352 includes a base structure 354having a stand 356. The transducer 314 is slidably mounted to the basestructure 354 by a guide 364. The guide 364 is either integrally formedwith the base structure 354 or fixedly attached to the base structure.The support structure 352 also includes a holder 360 attached to thebase structure 354 for holding the container 358. The holder 360 has aU-shaped bottom portion providing access to the flexible wall 440 of thechamber 367. The guide 364 and the holder 360 are arranged to hold thetransducer 314 and the container 358, respectively, such that theexternal surface of the wall 440 contacts the transducer 314. Thesupport structure 352 also includes a top retainer 362 for the container358. The retainer 362 is U-shaped to allow access to an exit port 444formed in the container 358.

The support structure 352 further includes an elastic body, such as aspring 366, for applying a force to the transducer 314 to press thetransducer 314 against the wall 440. When the transducer 314 is incontact with the wall 440, the force provided by the spring 366 isconstant, providing for consistent coupling between the transducer 314and the wall 440. The spring 366 is positioned between a spring guide372 and the base of a coupler 368 that supports the bottom of thetransducer 314. As shown in FIG. 36, the coupler 370 preferably has awindow 370 through which the power cord (not shown) of the transducer314 may be placed. Bolts or screws 376 hold the spring guide 372 inadjustment grooves 374 formed in the base structure 354. The magnitudeof the force provided by the spring 366 may be adjusted by changing thepreload on the spring. To adjust the preload on the spring 366, thebolts 376 holding the spring guide 372 are loosened, the guide 372 ismoved to a new position, and the bolts 376 are retightened to hold theguide 372 in the new position. Once the preload on the spring 366 isadjusted to provide a suitable coupling force between the transducer 314and the wall 440, it is desirable to keep the preload constant from oneuse of the apparatus 350 to the next so that valid comparisons can bemade between different samples disrupted by the apparatus.

The magnitude of the force provided by the spring 366 to press togetherthe transducer 314 and the wall 440 is important for achieving aconsistent transfer of energy between the transducer 314 and the chamber367. If the force is too light, the transducer 314 will only be heldlightly against the wall 440, leading to poor translation of vibratorymovement from the transducer 314 to the wall 440. If the force is toostrong, the container 358 or wall 440 may be damaged during sonication.An intermediate force results in the most consistent and repeatabletransfer of vibratory motion from the transducer 314 to the wall 440. Itis presently preferred that the spring 366 provide a force in the rangeof 2 to 5 lbs.

FIG. 38 shows an exploded view of the container 358, and FIG. 39 showsan assembled view of the container 358. As shown in FIGS. 38-39, thecontainer 358 has a body comprising a top piece 448, a middle piece 450,and a bottom piece 452. The middle piece 450 defines an inlet port 442to the chamber 367, and the top piece 448 defines an outlet port 444 tothe chamber. The ports 442, 444 are positioned to permit the continuousflow of a fluid sample through the chamber 367. The flexible wall 440 isheld between the middle and bottom pieces 450, 452 using gaskets 453,454. Alternatively, the flexible wall 440 may simply be heat sealed tothe middle piece 450 so that the bottom piece 452 and gaskets 453, 454may be eliminated.

The container 358 also includes a filter stack 446 in the chamber 367for capturing sample components (e.g., target cells or viruses) as thesample flows through the chamber 367. The filter stack comprises (frombottom to top in FIGS. 38-39) a gasket 456, a first filter 458, a gasket460, a second filter 464 having a smaller average pore size than thefirst filter 458, and a gasket 466. The filter stack is held between thetop and middle pieces 448, 450 of the container 358. The filter stackalso includes beads 462 disposed between the first and second filters458 and 464. The gasket 460 spaces the first filter 458 from the secondfilter 464. The gasket 460 should be thick enough to permit the beads tomove freely in the space between the filters 458, 464. A fluid sampleflowing through the chamber 367 first flows through the filter 458 andthen through the filter 466. After flowing through the filter stack, thesample flows along flow ribs 468 (FIG. 38) formed in the portion of thetop piece 448 that defines the top of the chamber and through the outletport 444 (FIG. 39).

The filter stack is effective for capturing cells or viruses as a fluidsample flows through the chamber 367 without clogging of the. The firstfilter 458 (having the largest pore size) filters out coarse materialsuch as salt crystals, cellular debris, hair, tissue, etc. The secondfilter 464 (having a smaller pore size) captures target cells or virusesin the fluid sample. The average pore size of the first filter 458 isselected to be small enough to filter coarse material from the fluidsample (e.g., salt crystals, cellular debris, hair, tissue) yet largeenough to allow the passage of the target cells or viruses. In general,the average pore size of the first filter 458 should be in the range ofabout 2 to 25 μm, with a presently preferred pore size of about 5 μm.The average pore size of the second filter 464 is selected to beslightly smaller than the average size of the target cells or viruses tobe captured (typically in the range of 0.2 to 5 μm).

The beads 462 are useful for disrupting the captured cells or viruses torelease the intracellular material (e.g., nucleic acid) therefrom.Movement of the beads 462 ruptures the cells or viruses captured on thefilter 464. Suitable beads for rupturing cells or viruses includeborosilicate glass, lime glass, silica, and polystyrene beads. The beadsmay be porous or non-porous and preferably have an average diameter inthe range of 1 to 200 μm. In the presently preferred embodiment, thebeads 462 are polystyrene beads having an average diameter of about 100μm.

The beads 462 may have a binding affinity for target cells or viruses inthe fluid sample to facilitate capture of the target cells or viruses.For example, antibodies or certain receptors may be coated onto thesurface of the beads 462 to bind target cells in the sample. Moreover,the chamber 367 may contain two different types of beads for interactingwith target cells or viruses. For example, the chamber may contain afirst set of beads coated with antibodies or receptors for bindingtarget cells or viruses and a second set of beads (intermixed with thefirst set) for rupturing the captured cells or viruses. The beads in thechamber may also have a binding affinity for the intracellular material(e.g., nucleic acid) released from the ruptured cells or viruses. Suchbeads may be useful for isolating target nucleic acid for subsequentelution and analysis. For example, the chamber 367 may contain silicabeads to isolate DNA or cellulose beads with oligo dT to isolatemessenger RNA for RT-PCR. The chamber 367 may also contain beads forremoving unwanted material (e.g., proteins, peptides) or chemicals(e.g., salts, metal ions, or detergents) from the sample that mightinhibit PCR.

To ensure that the air bubbles can escape from the chamber 367, it isdesirable to use the container 358 in an orientation in which liquidflows up (relative to gravity) through the filters 458, 464 and thechamber 367. The upward flow through the chamber 367 aids the flow ofair bubbles out of the chamber. Thus, the inlet port 442 for entry offluids into the chamber 367 should generally be at a lower elevationthan the outlet port 444. The volume capacity of the chamber 367 isusually in the range of 50 to 500 μl. The volume capacity of the chamber367 is selected to provide for concentration of analyte separated from afluid sample without the chamber being so small that the filters 458,464 become clogged.

The pieces 448, 450, 452 forming the body of the container 358 arepreferably molded polymeric parts (e.g., polypropylene, polycarbonate,acrylic, etc.). Although molding is preferred for mass production, italso possible to machine the top, middle, and bottom pieces 448, 450,452. The pieces 448, 450, 452 may be held together by screws orfasteners. Alternatively, ultrasonic bonding, solvent bonding, or snapfit designs could be used to assemble the container 358. Another methodfor fabricating the container 358 is to mold the body as a single pieceand heat seal the flexible wall 440 and the filters 458, 464 to thebody.

FIG. 40 shows a fluidic system for use with the apparatus. The systemincludes a bottle 470 for holding lysis buffer, a bottle 472 containingwash solution, and a sample container 474 for holding a fluid sample.The bottles 470, 472 and sample container 474 are connected via tubingto the valve ports of a syringe pump 476. The inlet port of thecontainer 358 is also connected to the syringe pump 476. The outlet portof the container 358 is connected to the common port of a distributionvalve 478. The system also includes a collection tube 480 for receivingintracellular material removed from the sample, a waste container 482for receiving waste, and a pressure source, such as a pump 484. Thecollection tube 480, waste container 482, and pump 484 are connected torespective peripheral ports of the distribution valve 478. A pressureregulator 486 regulates the pressure supplied by the pump 484.

A specific protocol will now be described with reference to FIGS. 39-40to illustrate the operation of the container 358. It is to be understoodthat this is merely an example of one possible protocol and is notintended to limit the scope of the invention. The syringe pump 476 pumpsa fluid sample from the sample container 474 through the container 358and into the waste container 482. As the fluid sample is forced to flowthrough the filters in the chamber 367, coarse material is filtered bythe filter 458 and target cells or viruses in the sample are captured bythe filter 464. The chamber 367 may be sonicated as the sample is forcedto flow through the chamber to help prevent clogging of the filters.Next, the syringe pump 476 pumps wash solution from the bottle 472through the container 358 and into the waste container 482. The washingsolution washes away PCR inhibitors and contaminants from the chamber367.

In the next step, the syringe pump 476 pumps lysis buffer from thebottle 470 into the container 358 so that the chamber 367 is filled withliquid. The lysis buffer should be a medium through which dynamicpressure pulses or pressure waves can be transmitted. For example, thelysis buffer may comprise deionized water for holding the cells orviruses in suspension or solution. Alternatively, the lysis buffer mayinclude one or more lysing agents to aid in the disruption of the cellsor viruses. One of the advantages of the present invention, however, isthat harsh lysing agents are not required for successful disruption ofthe cells or viruses. Next, the distribution valve of the syringe pump476 is closed upstream of the container 358, and the distribution valve478 is opened. The pump 484 then pressurized the chamber 367 through theoutlet port 444, preferably to about 20 psi above the ambient pressure.The distribution valve 478 downstream of the container 358 is thenclosed. The static pressure in the chamber 367 is therefore increased toabout 20 psi in preparation for the disruption of the cells or virusestrapped on the filter 464.

Referring again to FIG. 37, the pressurization of the chamber 367 isimportant because it ensures effective coupling between the transducer314 and the flexible wall 440. To disrupt the cells or viruses in thechamber 367, the transducer 314 is activated (i.e., set into vibratorymotion). The flexible wall 440 transfers the vibrational motion of thetransducer 314 to the liquid in the chamber 367 by allowing slightdeflections without creating high stresses in the wall. The transducer314 is preferably an ultrasonic horn for sonicating the chamber 367. Thechamber 367 is preferably sonicated for 10 to 40 seconds at a frequencyin the range of 20 to 60 kHz. In the exemplary protocol, the chamber issonicated for 15 seconds at a frequency of 40 kHz. The amplitude of thehorn tip is preferably in the range of 20 to 25 μm (measured peak topeak).

As the tip of the transducer 314 vibrates, it repeatedly impacts theflexible wall 440. On its forward stroke (in the upward direction inFIG. 37), the tip of the transducer 314 pushes the wall 440 and createsa pressure pulse or pressure wave in the chamber 367. On its retreatingstroke (downward in FIG. 37), the tip of the transducer 314 usuallyseparates from the flexible wall 440 because the flexible wall 440cannot move at the same frequency as the transducer. On its next forwardstroke, the tip of the transducer 314 once again impacts the wall 440 ina head-on collision as the tip and wall speed towards each other.Because the transducer 314 and the wall 440 separate as the transducer314 vibrates, the effective forward stroke of the transducer is lessthan its peak-to-peak amplitude. The effective forward stroke determinesthe level of sonication in the chamber 367. It is therefore important toincrease the static pressure in the chamber 367 so that when the tip ofthe transducer 314 retreats, the flexible wall 440 is forced outwardlyto meet the tip on its return stroke. The static pressure in the chamber367 should be sufficient to ensure that the effective forward stroke ofthe transducer 314 generates the necessary pressure pulses or pressurewaves in the chamber to effect cell disruption. It is presentlypreferred to increase the static pressure in the chamber 367 to at least5 psi above the ambient pressure, and more preferably to a pressure inthe range of 15 to 25 psi above the ambient pressure.

On each forward stroke, the transducer 314 imparts a velocity to theliquid in the chamber 367, thus creating a pressure pulse or pressurewave that quickly sweeps across the chamber. The beads 462 in the filterstack 446 (FIG. 38) are agitated by the pressure pulses in the chamber367. The pressure pulses propel the beads into violent motion, and thebeads mechanically rupture the cells or viruses to release the analyte(e.g., nucleic acid) therefrom. Referring again to FIG. 40, followingdisruption of the cells or viruses, the syringe pump 476 pumps thereleased intracellular material from the container 358 into thecollection tube 480.

FIG. 41 shows another embodiment of the invention in which the container358 has a solid wall 488 for contacting the transducer 314. The solidwall 488 differs from the flexible wall 440 previously described withreference to FIG. 37. Whereas the flexible wall is typically a thin filmthat bends under its own weight and does not hold its shape unless heldon its edges, the solid wall 488 holds it shape when unsupported. Theadvantage of using a solid wall to contact the transducer 314 is thatthere is no need to pressurize the chamber 367 to ensure effectivecoupling between the wall 488 and the transducer 314. The elasticrestoring force of the solid wall 488 provides the necessary couplingbetween the wall and the transducer 314. However, the proper design ofthe solid wall 488 is necessary so that the wall is not damaged (e.g.,melted) by the vibratory movements of the transducer 314.

In particular, the solid wall 488 should have a natural frequency thatis higher than the vibrating frequency at which the transducer 314 isoperated. Preferably, the ratio of the natural frequency of the wall 488to the vibrating frequency is at least 2:1, and more preferably theratio is at least 4:1. In addition, the wall 488 should not be so rigidthat it cannot transfer the vibratory motion of the transducer to theliquid in the chamber 367. It is preferred that the wall 488 be capableof deflecting a distance in the range of 5 to 40 μm, and more preferablyabout 20 μm peak to peak when the transducer 314 applies a force in therange of 1 to 10 lbs. to the external surface of the wall 488. It ismore preferable that the wall 488 be capable of deflecting a distance inthe range of 5 to 40 μm, and more preferably about 20 μm peak to peakwhen the transducer 314 applies a force in the range of 2 to 5 lbs. Toachieve these criteria, the wall 488 is dome-shaped and convex withrespect to the transducer 314 (i.e., the wall 488 curves outwardlytowards the transducer). The advantage to the dome-shaped design of thewall 488 is that the dome shape increases the natural frequency of thewall (compared to a flat wall) without causing the wall to be so stiffthat it cannot transfer the vibratory movements of the transducer 314 tothe chamber 367.

FIG. 42 shows a cross sectional view of the wall 488. The dome-shapedportion 495 of the wall preferably has a radius of curvature R in therange of 6.3 to 12.7 mm when the diameter D of the dome-shaped portionis about 11.1 mm. More preferably, the dome-shaped portion 495 of thewall preferably has a radius of curvature R of about 9.5 mm when thediameter D of the dome-shaped portion is about 11.1 mm. The wall 488also includes a flat outer rim 497 for clamping the wall 488 in thecontainer 358. Alternatively, the wall 488 may be integrally molded witheither of pieces 450, 452 (FIG. 41). The thickness T of the wall ispreferably in the range of 0.25 to 1 mm. If it is less than 0.25 mmthick, the wall 488 may be too weak. If the wall has a thickness greaterthan 1 mm, the wall may be too stiff to deflect properly in response tothe vibratory movements of the transducer. In the presently preferredembodiment, the wall 488 has a thickness T of about 0.5 mm. The wall 488is preferably a molded plastic part. Suitable materials for the wall 488include Delrin® (acetal resins or polymethylene oxide), polypropylene,or polycarbonate.

The interaction of the transducer 314 with the solid wall 488 will nowbe described with reference to FIG. 41. Prior to activating thetransducer, target cells or viruses are captured on the filter 490 byforcing a fluid sample to flow though the chamber 367 (e.g., using thefluidic system previously described with reference to FIG. 40). Inaddition, the chamber 367 is filled with a liquid (e.g., lysis buffer)as previously described. Unlike the previously described embodiments,however, the chamber 367 does not require pressurization. Instead, it ispreferred that ambient pressure is maintained in the chamber. Thetransducer 314 is placed in contact with the external surface of thewall 488, preferably using a support structure as previously describedwith reference to FIG. 37. In particular, a spring preferably pushes thetransducer against the wall 488 with a force in the range of 1 to 10lbs., and more preferably in the range of 2 to 5 lbs.

To disrupt the cells or viruses in the chamber 367, the transducer 314is activated (i.e., induced into vibratory motion). As the tip of thetransducer 314 vibrates, it deflects the wall 488. On its forward stroke(in the upward direction in FIG. 41), the tip of the transducer 314pushes the wall 488 and creates a pressure pulse or pressure wave in thechamber 367. On its retreating stroke (downward in FIG. 41), the wall488 remains in contact with the tip of the transducer 314 because thewall 488 has a natural frequency higher than the vibrating frequency ofthe transducer. In embodiments in which the transducer is an ultrasonichorn for sonicating the chamber 367, the chamber 367 is preferablysonicated for 10 to 40 seconds at a frequency in the range of 20 to 40kHz. In the exemplary protocol, the chamber is sonicated for 15 secondsat a frequency of 40 kHz. The amplitude of the horn tip is preferably inthe range of 20 to 25 μm (measured peak to peak), and the naturalfrequency of the wall 488 should be greater than 40 kHz, preferably atleast 80 kHz, and more preferably at least 160 kHz.

One advantage to using the solid interface wall 488 is that strongpressure drops can be achieved in the chamber 367 as long as the staticpressure in the chamber is low. For example, at atmospheric pressure,cavitation (the making and breaking of microscopic bubbles) can occur inthe chamber 367. As these bubbles or cavities grow to resonant size,they collapse violently, producing very high local pressure changes. Thepressure changes provide a mechanical shock to the cells or viruses,resulting in their disruption. The disruption of the cells or virusesmay also be caused by sharp pressure rises resulting from the vibratorymovement of the transducer 314. In addition, the disruption of the cellsor viruses may be caused by the violent motion of the beads 462 in thechamber 367. The beads are agitated by the dynamic pressure pulses inthe chamber and rupture the cells or viruses. In experimental testing,the applicants have found that it is usually necessary to use beads todisrupt certain types of cells (particularly spores) having highlyresistant cell walls. Other types of cells, such as blood cells, areeasier to disrupt and may often be disrupted without the use of thebeads 462.

Although the use of an ultrasonic transducer has been described as apreferred embodiment, it is to be understood that different types oftransducers may be employed in the practice of the present invention.The transducer should be capable of creating pressure pulses or pressurewaves in the chamber 367. In addition, the transducer should be capableof providing high velocity impacts to the liquid in the chamber.Suitable transducers include ultrasonic, piezoelectric,magnetostrictive, or electrostatic transducer. The transducer may alsobe an electromagnetic device having a wound coil, such as a voice coilmotor or a solenoid device. The vibrating frequency of the transducermay be ultrasonic (i.e., above 20 kHz) or below ultrasonic (e.g., in therange of 60 to 20,000 Hz). The advantage to using higher frequencies isthat cell disruption is very rapid and can often be completed in 10 to20 seconds. The disadvantage is that ultrasonic transducers are oftenmore expensive than a simple mechanical vibrator, e.g., a speaker orelectromagnetic coil device. In one alternative embodiment, for example,the solid wall 488 is used in combination with a speaker orelectromagnetic coil device that vibrates at an operating frequency inthe range of 5 to 10 kHz.

FIGS. 43A-43B illustrate another solid wall 500 for contacting atransducer according to the present invention. As shown in FIG. 43A, oneside of the wall 500 has a central portion 502 and a plurality ofstiffening ribs 504 extending radially from the central portion 502. Thewall also has recesses 506 formed between the ribs 504. As shown in FIG.43B, the other side of the wall 500 has a flat surface 508. FIG. 44shows a partially-cut away isometric view of the container 358 with thewall 500. The wall 500 is preferably positioned so that the side of thewall having the flat surface is internal to the chamber 367 and suchthat the side of the wall having the ribs 504 is external to thechamber. The ribs 504 are advantageous because they increase the naturalfrequency of the wall without causing the wall to be so stiff that itcannot transfer the vibratory movements of the transducer to the chamber367.

FIG. 45 shows a bottom plan view of the container 358 having the wall500. The central portion 502 provides the external surface of the wall500 for contacting a transducer. The interaction of the wall 500 withthe transducer is analogous to the interaction of the wall 488 with thetransducer previously described with reference to FIG. 41. Inparticular, the wall 500 remains in contact with the tip of thetransducer because the wall 500 has a natural frequency higher than thevibrating frequency of the transducer. Consequently, pressurization isnot required, and cavitation may be achieved. The solid walls 488, 500described with reference to FIGS. 41-45 may be used in the container 358or the walls 488, 500 may be used in a fully integrated cartridge, suchas the cartridge shown in FIG. 1.

FIG. 46 shows a partially exploded view of a container 274 for holdingcells or viruses to be disrupted according to another embodiment of theinvention. FIG. 47 shows a front view of the container 274. As shown inFIGS. 46-47, the container 274 has a chamber 277 for holding a liquidcontaining cells or viruses to be disrupted. The container 274 has arigid frame 278 that defines the side walls 282A, 282B, 282C, 282D ofthe chamber 277. The rigid frame 278 also defines a port 276 and achannel 288 that connects the port 276 to the chamber 277. The containeralso includes thin, flexible sheets attached to opposite sides of therigid frame 278 to form two spaced-apart, opposing major walls 280A,280B of the chamber. The flexible major walls 280A, 280B are shown inFIG. 46 exploded from the rigid frame 278 for illustrative clarity. Whenthe container 274 is assembled, the major walls 280A, 280B are sealed toopposite sides of the frame 278, as is described in detail below. Thechamber 277 is thus defined by the spaced apart, opposing major walls280A, 202B and by the rigid side walls side walls 282A, 282B, 282C, 282Dthat connect the major walls to each other.

The container 274 also includes a plunger 284 that is inserted into thechannel 288 after adding the cells or viruses to the chamber 277. Theplunger 284 compresses gas in the container 274 thereby increasingpressure in the chamber 277. The gas compressed by the plunger 284 istypically air filling the channel 288. The pressurization of the chamber277 forces the flexible wall 280A to conform to the surface of thetransducer (not shown in FIGS. 46-47), as is discussed in greater detailbelow. The plunger 284 also closes the port 276 and seals the chamber277 from the environment external to the container.

In general, the plunger may comprise any device capable of establishinga seal with the walls of the channel 288 and of compressing gas in thecontainer. Such devices include, but are not limited to, pistons, plugs,or stoppers. The plunger 284 of the preferred embodiment includes a stem290 and a piston 292 on the stem. When the plunger 284 is inserted intothe channel 288, the piston 292 establishes a seal with the inner wallsof the channel and compresses air in the channel. The piston 292 ispreferably a cup integrally formed (e.g., molded) with the stem 290.Alternatively, the piston 292 may be a separate elastomeric pieceattached to the stem.

The plunger 284 also preferably includes an alignment ring 294encircling the stem for maintaining the plunger 284 in coaxial alignmentwith the channel 288 as the plunger is inserted into the channel. Thealignment ring 294 is preferably integrally formed (e.g., molded) withthe stem 290. The stem 290 may optionally includes support ribs 293 forstiffening and strengthening the stem. The plunger 284 also includes aplunger cap 296 attached to the stem 290. As shown in FIG. 47, the cap296 includes a snap ring 297 and the container includes an annularrecess 279 encircling the port 276 for receiving the snap ring 297. Thecap 296 may optionally include a lever portion 298 which is lifted toremove the plunger 284 from the channel 288. The container 274 may alsoinclude finger grips 287 for manual handling of the container.

FIG. 51 shows an isometric view of an apparatus 304 for disrupting cellsor viruses. The apparatus 304 includes a transducer 314, preferably anultrasonic horn, for generating pressure pulses the chamber of thecontainer 274. The apparatus 304 also includes a support structure 306for holding the transducer 314 and the container 274 against each other.The support structure 306 includes a base 308 and a first holder 310attached to the base for holding the outer housing of the transducer314. The holder 310 includes a bore for receiving the transducer 314 andscrews or bolts 312 that are tightened to clamp the outer housing of thehorn firmly in the holder. The base 308 may optionally include boltholes 320 for bolting the support structure 306 to a surface, e.g., acounter or bench top.

As shown in FIG. 52, the support structure 306 also includes a holder316 for holding the container 274. The holder 316 is slidably mounted tothe base 308 by means of a guide 318. The guide 318 may be fixedlyattached to the base 308 or integrally formed with the base. The guide318 has two guide pins 322, and the holder 316 has two guide slots 324for receiving the guide pins 322. The holder 316 may thus slide on theguide pins 322. As shown in the partially cut-away view of FIG. 53, theholder 316 is designed to hold the container 274 such that the externalsurface of the flexible wall 280A is exposed and accessible to the tip326 of the transducer 314. The guide 318 is appropriately aligned withthe transducer 314 to slide the holder 316 into a position in which theexternal surface of the flexible wall 280A contacts the tip 326.

FIG. 54 shows an isometric view of the holder 316. The holder 316 has abody 317 in which are formed the guide slots 324 for receiving the guidepins. The body also has a recess 334 for receiving the container 274.The shape of the recess 334 matches the shape of the lower portion ofthe frame 278 so that the frame fits securely in the recess 334. Theholder 316 also includes a retaining member 328 attached to the body 317by screws or bolts 330. The retaining member 328 and body 317 define aslot 332 through which the frame 278 is inserted when the frame isplaced in the recess 334. The retaining member 328 holds the frame 278in the recess. The body 317 also has an opening 336 adjacent the recess334. The shape of the opening 336 corresponds to the shape of thechamber 277.

As shown in the cross sectional view of FIG. 56, when the container 274is inserted into the holder 316, the opening 336 is positioned next tothe flexible wall 280B. The opening 336 is thus positioned to permit theflexible wall 280B to expand outwardly into the opening. The holder 316holds only the frame of the container 274 so that the flexible walls280A, 280B are unrestrained by the holder. The flexible wall 280A istherefore free to move inwardly and outwardly with the horn tip 326 asvibrational motion is transmitted from the tip 326 to the wall 280A. Theflexible wall 280B is also free to move inwardly or outwardly aspressure pulses sweep through the chamber 277. This permits the liquidwithin the chamber 277 to move more freely as it receives the dynamicpressure pulses and thus enhances the cell disruption in the chamber277. Venting of the opening 336 is provided by first and second bores338, 344 formed in the body of the holder 316. One end of the narrowerbore 338 is connected to the opening 336 and the other end is connectedto the larger bore 344. The bore 344 extends through the body of theholder 316 to permit the escape of gas (e.g., air) from the opening 336.The venting prevents pressure from building in the opening 336 when theflexible wall 280B expands into the opening. Such pressure wouldrestrict the motion of the wall 280B.

Referring again to FIG. 54, the container 274 has a bulb-shaped tab 275extending from the bottom of the frame 278. The holder 316 has holes 340formed in the body 317 adjacent the recess 334. When the frame 278 isinserted into the recess 334, the tab 275 is positioned between theholes 340. The holes 340 are for receiving retaining pins. As shown inFIG. 55, the retaining pins 342 extend from the guide 318 (from whichthe guide pins have been removed for clarity in FIG. 55) and arepositioned on opposite sides of the bulb-shaped tab 275 when thecontainer 274 is moved into contact with the horn tip 326. The spacingof the pins 342 is less than the width of the bulb so that the pins 342hold down the tab 275, and thus the container 274, as ultrasonic energyis transmitted into the container from the transducer 314. This ensuresthat the container 274 does not rise out of position due to the motionof the horn tip 326. Alternatively, a collar or other suitable retentionmechanism may be used to hold the container 274 in position.

Referring to FIG. 56, the support structure 306 also includes an elasticbody, such as a spring 366, for applying a force to the holder 316 topress the wall 280A of the chamber 277 against the horn tip 326. Whenthe wall 280A is in contact with the horn tip 326, the force provided bythe spring is constant, providing for consistent coupling and transferof power between the transducer 314 and the container 274. The spring366 is positioned in the bore 344. The holder 316 has an inner surfacesurrounding the junction of the larger bore 344 and the narrower bore338. One end of the spring 366 contacts the inner surface, and the otherend of the spring contacts a rod 348 that extends from the guide 318.The spring 366 is thus compressed between the surface of the holder 316and the rod 348 so that it pushes the holder 316, and thus the flexiblewall 280A of the container 274, against the tip 326.

The magnitude of the force provided by the spring 366 may be adjusted bychanging the preload on the spring. The support structure 306 includes arod 348 that contacts one end of the spring. The guide 318 includes afirst bore for receiving the rod 348 and a second bore for receiving aset screw 349 that holds the rod 348 in a fixed position. To adjust thepreload on the spring 366, the screw 349 is loosened, the rod 348 ismoved to a new position, and the screw 349 is retightened to hold therod 348 in the new position. The rod 348 and set screw 349 thus providea simple mechanism for adjusting the preload on the spring 366. Once thepreload on the spring 366 is adjusted to provide a suitable couplingforce between the wall 280A and the horn tip 326, it is desirable tokeep the preload constant from one use of the apparatus to the next sothat valid comparisons can be made between different samples disruptedby the apparatus.

The flexible wall 280A facilitates the transfer of vibrating motion fromthe transducer 314 to the chamber 277. The wall 280A is sufficientlyflexible to conform to the surface of the tip 326 of the transducer,ensuring good coupling between the tip 326 and the wall 280A. Thesurface of the tip 326 that contacts the wall 280A is preferably planar(e.g., flat) to ensure power coupling over the entire area of thesurface. Alternatively, the tip 326 may have a slightly curved (e.g.,spherical) surface for contacting the wall 280A. The opposite wall 280Bis preferably sufficiently flexible to move inwardly and outwardly asdynamic pressure pulses are generated in the chamber 277. This permitsthe liquid within the chamber 277 greater freedom of movement as itreceives the pressure pulses and thus enhances the action in the chamber277.

Referring again to FIG. 46, the walls 280A, 280B are preferably flexiblesheets or films of polymeric material such as polypropylene,polyethylene, polyester, or other polymers. The films may either belayered, e.g., laminates, or the films may be homogeneous. Layered filmsare preferred because they generally have better strength and structuralintegrity than homogeneous films. Alternatively, the walls 280A, 280Bmay comprise any other material that may be formed into a thin, flexiblesheet. For good flexibility and energy transfer, the thickness of eachwall is preferably in the range of 0.01 to 0.2 mm, and more preferablyin the range of 0.025 to 0.1 mm. As previously described, the plunger284 is inserted into the channel 288 after adding the cells or virusesto the chamber 277. The plunger 284 compresses air in the channel 288,thereby increasing pressure in the chamber 277. The pressurization ofthe chamber 277 ensures effective coupling between the wall 280A and thetip of the transducer 314.

It is presently preferred to pressurize the chamber 277 to a pressure inthe range of 2 to 50 psi above ambient pressure. This range is presentlypreferred because 2 psi is generally enough pressure to ensure effectivecoupling between the flexible wall 280A and the transducer 314, whilepressures above 50 psi may cause bursting of the walls 280A, 280B ordeformation of the frame of the container 274. More preferably, thechamber 277 is pressurized to a pressure in the range of 8 to 15 psiabove ambient pressure. This range is more preferred because it issafely within the practical limits described above.

A preferred method for disrupting cells or viruses using the apparatus304 will now be described with reference to FIGS. 46-56. Referring toFIG. 50, beads 301 are placed in the chamber 277 of the container toenhance the disruption of the cells or viruses. In general, the beads301 may be composed of glass, plastic, polystyrene, latex, crystals,metals, metal oxides, or non-glass silicates. The beads 301 may beporous or non-porous and preferably have a diameter in the range of 1 to200 μm. More preferably, the beads 301 are either polystyrene beads,borosilicate glass beads, or soda lime glass beads having an averagediameter of about 100 μm. The beads 301 may be placed in the chamber 277using a funnel. The funnel should be sufficiently long to extend fromthe port 276 through the channel 288 and into the chamber 277. Afterinserting the funnel into the container 274, the beads 301 are placed inthe funnel and the container 274 is tapped lightly (e.g., against abench top) until the beads 301 settle into the bottom of the chamber277. It is preferred that the funnel extend through the channel 288 andinto the chamber 277 as the beads 301 are added to the chamber toprevent the beads from contaminating the channel. The presence of beadsin the channel 288 would interfere with the subsequent stroke of theplunger into the channel. The quantity of beads 301 added to the chamber277 is preferably sufficient to fill about 10% to 40% of the volumecapacity of the chamber. For example, in the presently preferredembodiment, the chamber 277 has a volume capacity of about 100 □l, and30 to 40 mg of beads are placed into the chamber.

After the beads 301 are placed in the chamber 277, the chamber is filledwith a liquid containing the cells or viruses to be disrupted. Thechamber 277 may be filled using a pipette having a pipette tip 300(e.g., a standard 200 μl loading tip). Alternatively, the chamber 277may be filled using a syringe or any other suitable injection system.The liquid should be a medium through which pressure waves or pressurepulses can be transmitted. For example, the liquid may comprisedeionized water or ultrasonic gel for holding the cells or viruses insuspension or solution. Alternatively, the liquid may comprise abiological sample containing the cells or viruses. Suitable samplesinclude bodily fluids (e.g., blood, urine, saliva, sputum, seminalfluid, spinal fluid, mucus, etc) or environmental samples such as groundor waste water. The sample may be in raw form or mixed with diluents orbuffers. The liquid or gel may also include one or more lysing agents toaid in the disruption of the cells or viruses. One of the advantages ofthe present invention, however, is that harsh lysing agents are notrequired for successful disruption of the cells or viruses.

After the container 274 is filled with the liquid, the plunger 284 isinserted into the channel 288 to seal and pressurize the container 274.As the plunger 284 is inserted, the piston 292 compresses gas in thechannel 288 to increase pressure in the chamber 277, preferably to about8 to 15 psi above ambient pressure, as previously described.

Referring to FIG. 56, the holder 316 is then pushed or pulled away fromthe tip 326 of the transducer 314 (in the direction of the rod 348) sothat the container 274 can be inserted into the holder. The container274 is then placed in the holder 316. During the insertion of thecontainer 274, the holder 316 should be held a sufficient distance fromthe retaining pins 342 to provide clearance between the pins 342 and thetab 275. After the container 274 is inserted into the holder 316, theholder is gently released and the spring 366 pushes the holder 316 alongthe guide 318 until the wall 280A contacts and conforms to the surfaceof the horn tip 326. When the wall 280A is coupled to the horn tip 326,the spring 366 applies to the holder 316, and thus to the container 274,a substantially constant force to press the wall 280A against the horntip 326. The force provided by the spring 366 ensures effective couplingbetween the wall 280A and horn tip 326 as energy is transmitted to thechamber 277. As shown in FIG. 55, when the container 274 is moved intocontact with the tip 326, the tab 275 slides between the retaining pins342. The pins 342 prevent the container from sliding upward in responseto the vibratory motion of the tip 326.

Referring again to FIG. 56, the cells or viruses in the chamber 277 arethen disrupted by the pressure pulses and resulting bead movement in thechamber 277 generated by the vibration of the tip 326 against theflexible wall 280A. The magnitude of the force provided by the spring366 to press together the wall 280A and the tip 326 is important forachieving a consistent transfer of energy between the transducer 314 andthe chamber 277. If the force is too light, the wall 280A will only beheld lightly against the tip 326, leading to poor transmission of thevibratory movement of the transducer 314. If the force is too strong,the container 274 or wall 280A may be damaged during sonication. Anintermediate force results in the most consistent and repeatabletransfer of energy from the transducer 314 to the chamber 277. It ispresently preferred that the spring 366 provide a force in the range of0.25 to 4 lbs., with a force of about 1 lb. being the most preferred.

When the transducer 314 is activated, the tip 326 vibrates to transmitultrasonic energy into the chamber 277. There is a relationship betweenthe coupling force between the wall 280A and the tip 326 and the desiredamplitude of the vibratory movements of the tip 326. A balance can besought between the coupling force and the amplitude. Generally, a lightcoupling force requires a greater amplitude to effect disruption of thecells or viruses, while a stronger coupling force requires lessamplitude to effect disruption. For the range of coupling forcespresently preferred (0.25 to 4 lbs.), the peak-to-peak amplitude of thevibratory movements should be in the range of 4 to 40 μm, with apreferred peak-to-peak amplitude of amount 15 μm.

Ultrasonic waves are preferably transmitted to the chamber 277 at afrequency in the range of 20 to 50 kHz, with a frequency of about 40 kHzbeing preferred. The duration of time for which the chamber 277 issonicated is preferably in the range of 5 to 30 seconds. This range ispreferred because it usually takes at least 5 seconds to disrupt thecells or viruses in the chamber, while sonicating the chamber for longerthan 30 seconds will most likely denature or shear the nucleic acidreleased from the disrupted cells or viruses. Extensive shearing of thenucleic acid could interfere with subsequent amplification or detection.More preferably, the chamber is sonicated for about 10-20 seconds tofall safely within the practical limits stated above. The optimal timethat a particular type of cell sample should be subjected to ultrasonicenergy may be determined empirically.

Following disruption of the cells or viruses, the container 274 isremoved from the holder 316 by pulling the holder 316 away from the tip326 and withdrawing the container from the holder. The liquid or gelcontaining the disrupted cells and released nucleic acid is then removedfrom the container 274. This may be accomplished by centrifuging thecontainer 274 and removing the supernatant using, e.g., a pipette orsyringe. Alternatively, the liquid may be removed from the container 274by setting the container on edge and at an incline until the beadsprecipitate. The beads usually settle in about 15 to 20 seconds. Whenthe beads have settled, the plunger is withdrawn from the container 274and the liquid is removed using a syringe or pipette. The releasednucleic acid contained in the liquid may then be amplified and detectedusing techniques well known in the art.

One advantage of the apparatus and method of the present invention isthat it provides for the rapid and effective disruption of cells orviruses, including tough spores, without requiring the use of harshchemicals. In addition, the apparatus and method provide for highlyconsistent and repeatable lysis of cells or viruses, so that consistentresults are achieved from one use of the apparatus to the next. Theamount of energy that is absorbed by the liquid and beads held in thechamber 277 depends on the amplitude of the oscillations of the tip 326,the mass of the contents of the chamber 277, the pressure in the chamber277, and the coupling force between the tip 326 and the wall 280A. Allfour of these parameters should be held substantially constant from oneuse of the apparatus to the next in order to achieve the same amount ofdisruption repeatably.

Many different modifications to the apparatus shown in FIG. 56 arepossible. For example, the holder 316 may be slidably mounted to thebase 308 by a variety of means, including rails, wheels, sliding in agroove, sliding in a cylinder, etc.

Alternatively, the holder 316 may be fixedly attached to the base 308and the transducer 314 slidably mounted to the base. In this embodiment,an elastic body is positioned to apply a force to the transducer 314(either directly or to a holder holding the horn) to press together thehorn tip 326 and the wall 280A. In addition, in each of theseembodiments, the elastic body may be positioned to either push or pullthe transducer 314 or the container 274 towards each other. For example,the spring 366 may be positioned to push or pull the holder 316 towardsthe horn tip 326 or to push or pull the transducer 314 towards theholder 316. Further, multiple elastic bodies may be employed to applyforces to both the container 274 and the transducer 314 to push or pullthem towards each other. All of these embodiments are intended to fallwithin the scope of the present invention.

Although a coil spring 366 is shown in FIGS. 37 and 56, it is to beunderstood that any type of elastic body may be used. Suitable elasticbodies include, but are not limited to, coil springs, wave springs,torsion springs, spiral springs, leaf spring, elliptic springs,half-elliptic springs, rubber springs, and atmospheric springs. Theelastic body may also be compressed air or rubber. Preferably, theelastic body is a coil spring. Coil springs are preferred because theyare simple and inexpensive to place in the apparatus and because thehave a low spring rate. A compressed air system is also effective, butconsiderably more expensive. In embodiments in which the elastic body isa spring, the spring should have a low spring rate, preferably less than4 lb./in. A low spring rate minimizes the effect that any variations inthe thickness of the chamber 277 (due to small variations inmanufacturing, filling, or pressurizing the container) will have on themagnitude of the force provided by the spring to press together the wall280A and the horn tip 326.

Another advantage of the container 274 is that the chamber 277 holds thecells or viruses in a thin volume of liquid that can be uniformlysonicated easily. Referring to FIGS. 48-49, it is presently preferred toconstruct the container 274 such that each of the sides walls 282A,282B, 282C, 282D of the chamber has a length L in the range of 5 to 20mm, the chamber has a width W in the range of 7 to 30 mm, and thechamber has a thickness T in the range of 0.5 to 5 mm. In addition, thechamber 277 preferably has a width W greater than its thickness T. Inparticular, the ratio of the width W of the chamber to the thickness Tof the chamber is preferably at least 2:1. More preferably, the ratio ofthe width W of the chamber to the thickness T of the chamber is at least4:1. These ratios are preferred to enable the entire volume of thechamber 277 to be rapidly and uniformly sonicated. In general, thevolume capacity of the chamber 277 is preferably in the range of 0.02 to1 ml.

Referring again to FIG. 56, the transducer 314 is preferably anultrasonic horn. The thickness of the chamber 277 (and thus the spacingbetween the walls 280A and 280B) is preferably less than half of thediameter of the tip 326 of the horn. This relationship between thethickness of the chamber 277 and the diameter of the tip 326 ensuresthat the ultrasonic energy received from the transducer 314 issubstantially uniform throughout the volume of the chamber 277. As aspecific example, in the presently preferred embodiment, the tip 326 hasa diameter of 6.35 mm and the chamber 277 has a thickness of about 1.0mm. In addition, the major wall 280A should be slightly larger than thesurface of the horn tip 326 that presses against the wall 280A. Thisallows the flexible wall 280A to flex in response to the vibratorymotion of the horn tip 326.

A preferred method for fabricating the container 274 will now bedescribed with reference to FIGS. 46-47. The container 274 may befabricated by first molding the rigid frame 278 using known injectionmolding techniques. The frame 278 is preferably molded as a single pieceof polymeric material, e.g., polypropylene or polycarbonate. After theframe 278 is produced, thin, flexible sheets are cut to size and sealedto opposite sides of the frame 278 to form the major walls 282A, 282B ofthe chamber 277.

The major walls 282A, 282B are preferably cast or extruded films ofpolymeric material, e.g., polypropylene films, that are cut to size andattached to the frame 278 using the following procedure. A first pieceof film is placed over one side of the bottom portion of the frame 278.The frame 278 preferably includes a tack bar 299 for aligning the topedge of the film. The film is placed over the bottom portion of theframe 278 such that the top edge of the film is aligned with the tackbar 299 and such that the film completely covers the bottom portion ofthe frame 278 below the tack bar 299. The film should be larger than thebottom portion of the frame 278 so that it may be easily held andstretched flat across the frame. The film is then cut to size to matchthe outline of the frame by clamping to the frame the portion of thefilm that covers the frame and cutting away the portions of the filmthat extend past the perimeter of the frame using, e.g., a laser or die.The film is then tack welded to the frame, preferably using a laser.

The film is then sealed to the frame 278, preferably by heat sealing.Heat sealing is presently preferred because it produces a strong sealwithout introducing potential contaminants to the container as the useof adhesive or solvent bonding techniques might do. Heat sealing is alsosimple and inexpensive. At a minimum, the film should be completelysealed to the surfaces of the side walls 282A, 282B, 282C, 282D. Morepreferably, the film is additionally sealed to the surfaces of thesupport ribs 295 and tack bar 299. The heat sealing may be performedusing, e.g., a heated platen. An identical procedure may be used to cutand seal a second sheet to the opposite side of the frame 278 tocomplete the chamber 277.

The plunger 284 is also preferably molded from polymeric material (e.g.,polypropylene or polycarbonate) using known injection moldingtechniques. As shown in FIG. 46, the frame 278, plunger 284, and leash286 connecting the plunger to the frame may all be formed in the samemold to form a one-piece part. This embodiment of the container isespecially suitable for manual use in which a human operator fills thecontainer and inserts the plunger 284 into the channel 288. The leash286 ensures that the plunger 284 is not lost or dropped on the floor.

The plunger 284 is presently preferred as a simple, effective, andinexpensive mechanism for increasing pressure in the chamber 277 and forsealing the chamber 277 from the external environment. It is to beunderstood, however, that the scope of the invention is not limited tothis embodiment. There are many other suitable techniques for sealingand pressurizing the container. In addition, any suitable pressuresource may be used to pressurize the chamber. Suitable pressure sourcesinclude syringe pumps, compressed air sources, pneumatic pumps, orconnections to external sources of pressure.

SUMMARY, RAMIFICATIONS, AND SCOPE

Although the above description contains many specificities, these shouldnot be construed as limitations on the scope of the invention, butmerely as examples of some of the presently preferred embodiments. Manymodifications or substitutions may be made to the apparatus and methodsdescribed without departing from the scope of the invention. Forexample, the container for holding the cells or viruses need not be oneof the specialized containers described in the various embodimentsabove. Any type of container having a chamber for holding the cells orviruses may be used to practice the invention. Suitable containersinclude, but are not limited to, reaction vessels, cuvettes, cassettes,and cartridges. The container may have multiple chambers and/or channelsfor performing multiple sample preparation functions, or the containermay have only a single chamber for holding cells or viruses fordisruption.

Further, the support structure for pressing the transducer and the wallof the container against each other may have many alternative forms. Forexample, in one alternative embodiment, the support structure includes avise or clamp for pressing the transducer and container against eachother. In another embodiment, the apparatus includes a pressure systemfor applying air pressure to press together the transducer and thecontainer. Alternatively, magnetic or gravitational force may be used topress together the transducer and the container. In each embodiment ofthe invention, force may be applied to the transducer, to the container,or to both the transducer and the container.

Therefore, the scope of the invention should be determined by thefollowing claims and their legal equivalents.

1-67. (canceled)
 68. An apparatus for disrupting cells or viruses, theapparatus comprising: a) a container having a chamber for holding thecells or viruses, wherein the container includes at least one walldefining the chamber; and b) a transducer for contacting an externalsurface of the wall and for vibrating at a frequency sufficient togenerate pressure waves in the chamber, wherein the natural frequency ofthe wall is greater than the vibrating frequency of the transducer. 69.The apparatus of claim 68, wherein the ratio of the natural frequency ofthe wall to the vibrating frequency of the transducer is at least 2:1.70. The apparatus of claim 68, wherein the ratio of the naturalfrequency of the wall to the vibrating frequency of the transducer is atleast 4:1.
 71. The apparatus of claim 68, wherein the wall issufficiently deformable to deflect a distance in the range of 5 to 40 μmwhen the transducer applies a force in the range of 1 to 10 lbs. to thewall.
 72. The apparatus of claim 68, wherein the wall is sufficientlydeformable to deflect a distance in the range of 5 to 40 μm when thetransducer applies a force in the range of 2 to 5 lbs. to the wall. 73.The apparatus of claim 68, wherein the wall is dome-shaped and convexwith respect to the transducer.
 74. The apparatus of claim 68, whereinthe wall includes stiffening ribs.
 75. The apparatus of claim 74,wherein the ribs extend radially from a central portion of the wall. 76.The apparatus of claim 68, wherein the transducer comprises anultrasonic horn.
 77. The apparatus of claim 68, further comprising beadsin the chamber for rupturing the cells or viruses.
 78. The apparatus ofclaim 68, further comprising a support structure for holding thecontainer and the transducer against each other such that the transducercontacts the external surface of the wall and for applying to thecontainer or to the transducer a substantially constant force to presstogether the transducer and the wall.
 79. A method for disrupting cellsor viruses, the method comprising the steps of: a) placing in thechamber of a container: i) the cells or viruses to be disrupted; and ii)a liquid; wherein the container includes at least one wall defining thechamber; b) generating pressure waves in the chamber by contacting anexternal surface of the wall with a vibrating transducer, wherein thenatural frequency of the wall is greater than the vibrating frequency ofthe transducer.
 80. The method of claim 79, further comprising the stepof agitating beads in the chamber.
 81. The method of claim 79, furthercomprising the step of applying to the container or to the transducer asubstantially constant force to press together the transducer and thewall.
 82. The method of claim 81, wherein the force is in the range of 2to 5 lbs.